Method for producing polymers having a preselected activity

ABSTRACT

The present invention relates to a method for isolating from the immunological gene repertoire a gene coding for a receptor having the ability to bind a preselected ligand. Receptors produced by the gene isolated by the method, particularly catalytic receptors, are also contemplated.

CROSS REFERENCE TO RELATED APPLICATION

This application is a divisional of U.S. application Ser. No.07/941,761, filed Nov. 25, 1992 now U.S. Pat. No. 6,291,160 which is acontinuation of U.S. application Ser. No. 07/799,772, filed Nov. 27,1991, abandoned; which is a continuation of U.S. application Ser. No.07/496,522, filed Mar. 20, 1990, abandoned which is acontinuation-in-part application of applications Ser. No. 07/411,058,having the same title and filed Sep. 21, 1989, abandoned which is acontinuation-in-part of Ser. No. 07/410,716, having the same title andfiled Sep. 20, 1989, abandoned which is a continuation-in-part of Ser.No. 07/352,884, having the same title and filed May 17, 1989, abandonedwhich is a continuation-in-part of Ser. No. 07/352,927, having the sametitle and filed May 16, 1989 abandoned. This is also acontinuation-in-part application of application Ser. No. 07/410,749having the same title and filed Sep. 20, 1989, abandoned which is acontinuation application of Ser. No. 07/352,884 having the same titleand filed May 17, 1989 abandoned.

DESCRIPTION

1. Technical Field

The present invention relates to a method for producing polymers havinga preselected activity.

2. Background

Binding phenomena between ligands and receptors play many crucial rolesin biological systems. Exemplary of such phenomena are the binding ofoxygen molecules to deoxyhemoglobin to form oxyhemoglobin, and thebinding of a substrate to an enzyme that acts upon it such as between aprotein and a protease like trypsin. Still further examples ofbiological binding phenomena include the binding of an antigen to anantibody, and the binding of complement component C3 to the so-calledCR1 receptor.

Many drugs and other therapeutic agents are also believed to bedependent upon binding phenomena. For example, opiates such as morphinere reported to bind to specific receptors in the brain. Opiate agonistsand antagonists are reported to compete with drugs like morphine forthose binding sites.

Ligands such as man-made drugs, like morphine and its derivatives, andthose that are naturally present in biological systems such asendorphins and hormones bind to receptors that are naturally present inbiological systems, and will be treated together herein. Such bindingcan lead to a number of the phenomena of biology, including particularlythe hydrolysis of amide and ester bonds as where proteins are hydrolyzedinto constituent polypeptides by an enzyme such as trypsin or papain orwhere a fat is cleaved into glycerine and three carboxylic acids,respectively. In addition, such binding can lead to formation of amideand ester bonds in the formation of proteins and fats, as well as to theformation of carbon to carbon bonds and carbon to nitrogen bonds.

An exemplary receptor-producing system in vertebrates is the immunesystem. The immune system of a mammal is one of the most versatilebiological systems as probably greater than 1.0×10⁷ receptorspecificities, in the form of antibodies, can be produced. Indeed, muchof contemporary biological and medical research is directed towardtapping this repertoire. During the last decade there has been adramatic increase in the ability to harness the output of the vastimmunological repertoire. The development of the hybridoma methodologyby Kohler and Milstein has made it possible to produce monoclonalantibodies, i.e., a composition of antibody molecules of a singlespecificity, from the repertoire of antibodies induced during an immuneresponse.

Unfortunately, current methods for generating monoclonal antibodies arenot capable of efficiently surveying the entire antibody responseinduced by a particular immunogen. In an individual animal there are atleast 5-10,000 different B-cell clones capable of generating uniqueantibodies to a small relatively rigid immunogens, such as, for exampledinitrophenol. Further, because of the process of somatic mutationduring the generation of antibody diversity, essentially an unlimitednumber of unique antibody molecules may be generated. In contrast tothis vast potential for different antibodies, current hybridomamethodologies typically yield only a few hundred different monoclonalantibodies per fusion.

Other difficulties in producing monoclonal antibodies with the hybridomamethodology include genetic instability and low production capacity ofhybridoma cultures. One means by which the art has attempted to overcomethese latter two problems has been to clone the immunoglobulin-producinggenes from a particular hybridoma of interest into a procaryoticexpression system. See, for example, Robinson et al., PCT PublicationNo. WO 89/0099; Winter et al., European Patent Publication No. 0239400;Reading, U.S. Pat. No. 4,714,681; and Cabilly et al., European PatentPublication No. 0125023.

The immunologic repertoire of vbertebrates has recently been found tocontain genes coding for immunoglobulins having catalytic activity.Tramontano et al., Sci., 234:1566-1570 (1986); Pollack et al., Sci.,234:1570-1573 (1986); Janda et al., Sci., 241:1188-1191 (1988); andJanda et al., Sci., 244:437-440 (1989). The presence of, or the abilityto induce the repertoire to produce, antibodies molecules capable of acatalyzing chemical reaction, i.e., acting like enzymes, had previouslybeen postulated almost 20 years ago by W. P. Jencks in Catalysis inChemistry and Enzymology, McGraw-Hill, N.Y. (1969).

It is believed that one reason the art failed to isolate catalyticantibodies from the immunological repertoire earlier, and its failure toisolate many to date even after their actual discovery, is the inabilityto screen a large portion of the repertoire for the desired activity.Another reason is believed to be the bias of currently availablescreening techniques, such as the hybridoma technique, towards theproduction high affinity antibodies inherently designed forparticipation in the process of neutralization, as opposed to catalysis.

BRIEF SUMMARY OF THE INVENTION

The present invention provides a novel method for screening a largerportion of a conserved receptor coding gene repertoire for receptorshaving a preselected activity than has heretofore been possible, therebyovercoming the before-mentioned inadequacies of the hybridoma technique.

In one embodiment, a conserved receptor-coding gene library containing asubstantial portion of the conserved receptor-coding gene repertoire issynthesized. In preferred embodiments, the conserved receptor-codinggene library contains at least about 10³, preferably at least about 10⁴and more preferably at least about 10⁵ different receptor-coding genes.

The gene library can be synthesized by either of two methods, dependingon the starting material.

Where the starting material is a plurality of receptor-coding genes, therepertoire is subjected to two distinct primer extension reactions. Thefirst primer extension reaction uses a first polynucleotide synthesisprimer capable of initiating the first reaction by hybridizing to anucleotide sequence conserved (shared by a plurality of genes) withinthe repertoire. The first primer extension produces of differentconserved receptor-coding homolog compliments (nucleic acid strandscomplementary to the genes in the repertoire).

The second primer extension reaction produces, using the complements astemplates, a plurality of different conserved receptor-coding DNAhomologs. The second primer extension reaction uses a secondpolynucleotide synthesis primer that is capable of initiating the secondreaction by hybridizing to a nucleotide sequence conserved among aplurality of the compliments.

Where the starting material is a plurality of compliments of conservedreceptor-coding genes, the repertoire is subjected to theabove-discussed second primer extension reaction. Of course, if both arepertoire of conserved receptor-coding genes and their complements arepresent, both approaches can be used in combination.

A conserved receptor-coding DNA homolog, i.e., a gene coding for areceptor capable of binding the preselected ligand, is then segregatedfrom the library to produce the isolated gene. This is typicallyaccomplished by operatively linking for expression a plurality of thedifferent conserved receptor-coding DNA homologs of the library to anexpression vector. The receptor-expression vectors so produces areintroduced into a population of compatible host cells, i.e., cellscapable of expressing a gene operatively linked for expression to thevector. The transformants are cultured under conditions for expressingthe receptor corded for by the receptor-coding DNA homolog. Thetransformants are cloned and the clones are screened for expression of areceptor that binds the preselected ligand. Any of the suitable methodswell known in the art for detecting the binding of a ligand to areceptor can be used. A transformant expressing the desired activity isthen segregated from the population to produce the isolated gene.

In another embodiment, the present invention contemplates a gene librarycomprising an isolated admixture of at least about 10³, preferably atleast about 10⁴ and more preferably at least 10⁵ conservedreceptor-coding DNA homologs, a plurality of which share a conservedantigenic determinant. Preferably, the homologs are present in a mediumsuitable for in vitro manipulation, such as water, phosphate bufferedsaline and the like, which maintains the biological activity of thehomologs.

A receptor having a preselected activity, preferably catalytic activity,produced by a method of the present invention, preferably a monomer ordimer as described herein, is also contemplated.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIG. 1 Illustrates a schematic diagram of the immunoglobulin moleculeshowing the principal structural features. The circled area on the heavychain represents the variable region (V_(H)), a polypeptide containing abiologically active (ligand binding) portion of that region, and a genecoding for that polypeptide, are produced by the methods of the presentinvention. Sequences L03, L35, L47 and L48 could not be classified intoany predefined subgroups.

FIG. 2A Diagrammatic sketch of an H chain of human IgG (IgG1 subclass).Numbering is from the N-terminus on the left to the C-terminus on theright. Note the presence of four domains, each containing an intrachaindisulfide bond (S—S) spanning approximately 60 amino acid residues. Thesymbol CHO stands for carbohydrate. The V region of the heavy (H) chain(V_(H)) resembles V_(L) in having three hypervariable CDR (not shown).

FIG. 2B Diagrammatic sketch of a human K chain (Panel 1). Numbering isfrom the N-terminus on the left to the C-terminus on the right. Note theintrachain disulfide bond (S—S) spanning about the same number of aminoacid residues in the V_(L) and C_(L) domains. Panel 2 shows thelocations of the complementarity-determining regions (CDR) in the V_(L)domain. Segments outside the CDR are the framework segments (FR).

FIG. 3 Amino acid sequence of the V_(H) regions of 19 mouse monoclonalantibodies with specificity for phosphorylcholine (SEQ ID NOS:1-19). Thedesignation HP indicates that the protein is the product of a hybridoma.The remainder are myeloma proteins. (From Gearhart et al., Nature,291:29, 1981.)

FIG. 4 Illustrates the results obtained from PCR amplification of mRNAobtained from the spleen of a mouse immunized with FITC. Lanes R17-R24correspond to amplification reactions with the unique 5′ primers (2-9,Table 1) and the 3′ primer (12, Table 1), R16 represents the PCRreaction with the 5′ primer containing inosine (10, Table 1) and 3′primer (12, Table 1). Z and R9 are the amplification controls; control Zinvolves the amplification of V_(H) from a plasmid (PLR2) and R9represents the amplification from the constant regions of spleen mRNAusing primers 11 and 13 (Table 1).

FIG. 5 Nucleotide sequences are clones form the cDNA library of the PCRamplified V_(H) regions in Lambda ZAP. The N-terminal 110 bases arelisted here and the underlined nucleotides represent CDR1 (complementarydetermining region) (SEQ ID NOS: 20-37).

FIG. 6 The sequence of the synthetic DNA insert inserted into Lambda ZAPto produce Lambda Zap II V_(H) (Panel A) and Lambda Zap V_(L) (Panel B)(SEQ ID NOS: 38-45) expression vectors. The various features requiredfor this vector to express the V_(H) and V_(L)-coding DNA homologsinclude the Shine-Dalgarno ribosome binding site, a leader sequence todirect the expressed protein to the periplasm as described by Mouva etal., J. Biol. Chem., 255:27, 1980, and various restriction enzyme sitesused to operatively link the V_(H) and V_(L) homologs to the expressionvector. The V_(H) expression-vector sequence also contains a shortnucleic acid sequence that codes for amino acids typically found invariable regions heavy chain (V_(H) Backbone). This V_(H) Backbone isjust upstream and in the proper reading as the V_(H) DNA homologs thatare operatively linked into the XhoI and SpeI. The V_(L) DNA homologsare operatively linked into the V_(L) sequence (Panel B) at the NcoI andSpeI restriction enzyme sites and thus the V_(H) Backbone region isdeleted when the V_(L) DNA homologs are operatively linked into theV_(L) vector.

FIG. 7 The major features of the bacterial expression vector Lambda ZapII V_(H) (V_(H)-expression vector) are shown. The synthetic DNA sequencefrom FIG. 6 is shown at the top along with the T₃ polymerase promoterfrom Lambda ZapII. The orientation of the insert in Lambda Zap II isshown. The V_(H) DNA homologs are inserted into the XhoI and SpeIrestriction enzyme sites. The V_(H) DNA are inserted into the XhoI andSpeI site and the read through transcription produces the decapeptideepitope (tag) that is located just 3′ of the cloning sites.

FIG. 8 The major features of the bacterial expression vector Lambda ZapII V_(L) (V_(L) expression vector) are shown. The synthetic sequenceshown in FIG. 6 is shown at the top along with the T₃ polymerasepromoter from Lambda Zap II. The orientation of the insert in Lambda ZapII is shown. The V_(L) DNA homologs are inserted into the phagemid thatis produced by the in vivo excision protocol described by Short et al.,Nucleic Acids Res., 16:7583-7600, 1988. The V_(L) DNA homologs areinserted into the Nco I and Spe I cloning sites of the phagemid.

FIG. 9 A modified bacterial expression vector Lambda Zap II V_(L)II.This vector is constructed by inserting this synthetic DNA sequence,(SEQ ID NO: 46)

TGAATTCTAAACTAGTCGCCAAGGAGACAGTCATAATGAATCGAACTTAAGATTTGATCAGCGGTTCCTCTGTCAGTATTACTT

ATACCTATTGCCTACGGCAGCCGCTGGATTGTTATTACTCGCTGTATGGATAACGGATGCCGTCGGCGACCTAACAATAATGAGCGAC

CCCAACCAGCCATGGCCGAGCTCGTCAGTTCTAGAGTTAAGCGGCCGGGGTTGGTCGGTACCGGCTCGAGCAGTCAAGATCTCAATTCGCCGGCAGCT

into Lambda Zap II that has been digested with the restriction enzymesSac I and Xho I. This sequence contains the Shine-Dalgarno sequence(Ribosome binding site), the leader sequence to direct the expressedprotein to the periplasm and the appropriate nucleic acid sequence toallow the V_(L) DNA homologs to the operatively linked into the SacI andXbaI restriction enzymes sites provided by this vector.

FIG. 10 The sequence of the synthetic DNA segment inserted into LambdaZap II to produce the lambda V_(L)II-expression vector. The variousfeatures and restriction endonuclease recognition sites are shown (SEQID NOS: 47-48).

FIG. 11 The vectors for expressing V_(H) and V_(L) separately and incombination are shown. The various essential components of these vectorsare shown. The light chain vector or V_(L) expression vector can becombined with the V_(H) expression vector to produce a combinatorialvector containing both V_(H) and V_(L) operatively linked for expressionto the same promoter.

FIG. 12 The labelled proteins immunoprecipitated from E. coli containinga V_(H) and a V_(L) DNA homolog are shown. In lane 1, the backgroundproteins immunoprecipitated from E. coli that do not contain a V_(H) orV_(L) DNA homolog are shown. Lane 2 contains the V_(H) proteinimmunoprecipitated from E. coli containing only a V_(H) DNA homolog. Inlanes 3 and 4, the commigration of a V_(H) protein a V_(L) proteinimmunoprecipitated from E. coli containing both a V_(H) and a V_(L) DNAhomolog is shown. In lane 5 the presence of V_(H) protein and V_(L)protein expressed from the V_(H) and V_(L) DNA homologs is demonstratedby the two distinguishable protein species. Lane 5 contains thebackground proteins immunoprecipitated by anti-E. coli antibodiespresent in mouse ascites fluid.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Nucleotide: a monomeric unit of DNA or RNA consisting of a sugar moiety(pentose), a phosphate, and a nitrogenous heterocyclic base. The base islinked to the sugar moiety via the glycosidic carbon (1′ carbon of thepentose) and that combination of base and sugar is a nucleoside. Whenthe nucleoside contains a phosphate group bonded to the 3′ or 5′position of the pentose it is referred to as a nucleotide.

Base Pair (bp): a partnership of adenine (A) with thymine (T), or ofcytosine (C) with guanine (G) in a double stranded DNA molecule. In RNA,uracil (U) is substituted for thymine.

Nucleic Acid: a polymer of nucleotides, either single or doublestranded.

Gene: a nucleic acid whose nucleotide sequence codes for a RNA orpolypeptide. A gene can be either RNA or DNA.

Complementary Bases: nucleotides that normally pair up when DNA or RNAadopts a double stranded configuration.

Complementary Nucleotide Sequence: a sequence of nucleotides in asingle-stranded molecule of DNA or RNA that is sufficientlycomplementary to that on another single strand to specifically hybridizeto it with consequent hydrogen bonding.

Conserved: a nucleotide sequence is conserved with respect to apreselected (reference) sequence if it non-randomly hybridizes to anexact complement of the preselected sequence.

Hybridization: the pairing of substantially complementary nucleotidesequences (strands of nucleic acid) to form a duplex or heteroduplex bythe establishment of hydrogen bonds between complementary base pairs. Itis a specific, i.e. non-random, interaction between two complementarypolynucleotide that can be competitively inhibited.

Nucleotide Analog: a purine or pyrimidine nucleotide that differsstructurally from a A, T, G, C, or U, but is sufficiently similar tosubstitute for the normal nucleotide in a nucleic acid molecule.

DNA Homolog: is a nucleic acid having a preselected conserved nucleotidesequence and a sequence coding for a receptor capable of binding apreselected ligand.

B. Methods

The present invention contemplates a method of isolating from arepertoire of conserved genes a gene coding for a receptor having apreselected activity, preferably a catalytic activity. The receptor canbe a polypeptide, an RNA molecule, such as a transfer RNA, an RNAdisplaying enzymatic activity, and the like. Preferably, the receptorwill be a polypeptide capable of binding a ligand, such as an enzyme,antibody molecule or immunologically active portion thereof, cellularreceptor, or cellular adhesion protein coded for by one of the membersof a family of conserved genes, i.e., genes containing a conservednucleotide sequence of at least about 10 nucleotides in length.

Exemplary conserved gene families are those coding for immunoglobulins,major histocompatibility complex antigens of class I or II, lymphocytereceptors, integrins and the like.

Immunoglobulins

The immunoglobulins, or antibody molecules, are a large family ofmolecules that include several types of molecules, such as IgD, IgG,IgA, IgM and IgE. The antibody molecule is typically comprised of twoheavy (H) and light (L) chains with both a variable (V) and constant (C)region present on each chain. Several different regions of animmunoglobulin contain conserved sequences useful for isolating animmunoglobulin repertoire. Extensive amino acid and nucleic acidsequence data displaying exemplary conserved sequences is compiled forimmunoglobulin molecules by Kabat et al., in Sequences of Proteins ofImmunological Interest, National Institutes of Health, Bethesda, Md.,1987.

The C region of the H chain defines the particular immunoglobulin type.Therefore the selection of conserved sequences as defined herein fromthe C region of the H chain results in the preparation of a repertoireof immunoglobulin genes having members of the immunoglobulin type of theselected C region.

The V region of the H or L chain typically comprises four framework (FR)regions each containing relatively lower degrees of variability thatincludes lengths of conserved sequences. The use of conserved sequencesfrom the FR1 and FR4 (J region) framework regions of the V_(H) chain isa preferred exemplary embodiment and is described herein in theExamples. Framework regions are typically conserved across several orall immunoglobulin types and thus conserved sequences contained thereinare particularly suited for preparing repertoires having severalimmunoglobulin types.

Major Histocompatibility Complex

The major histocompatibility complex (MHC) is a large genetic locus thatencodes an extensive family of proteins that include several classes ofmolecules referred to as class I, class II or class III MHC molecules.Paul et al., in Fundamental Immunology, Raven Press, NY, pp. 303-378(1984).

Class I MHC molecules are a polymorphic group of transplantationantigens representing a conserved family in which the antigen iscomprised of a heavy chain and a non-MHC encoded light chain. The heavychain includes several regions, termed the N, C1, C2, membrane andcytoplasmic regions. Conserved sequences useful in the present inventionare found primarily in the N, C1 and C2 regions and are identified ascontinuous sequences of “invariant residues” in Kabat et al., supra.

Class II MHC molecules comprise a conserved family of polymorphicantigens that participate in immune responsiveness and are comprised ofan alpha and a beta chain. The genes coding for the alpha and beta chaineach include several regions that contain conserved sequences suitablefor producing MHC class II alpha or beta chain repertoires. Exemplaryconserved nucleotide sequences include those coding for amino acidresidues 26-30 of the A1 region, residues 161-170 of the A2 region andresidues 195-206 of the membrane region, all of the alpha chain.Conserved sequences are also present in the B1, B2 and membrane regionsof the beta chain at nucleotide sequences coding for amino acid residues41-45, 150-162 and 200-209, respectively.

Lymphocyte Receptors and Cell Surface Antigens

Lymphocytes contain several families of proteins on their cell surfacesincluding the T-cell receptor, Thy-1 antigen and numerous T-cell surfaceantigens including the antigens defined by the monoclonal antibodiesOKT4 (leu3), OKUT5/8 (leu2), OKUT3, OKUT1 (leu1), OKT 11 (leu5) OKT6 andOKT9. Paul, supra at pp. 458-479.

The T-cell receptor is a term used for a family of antigen bindingmolecules found on the surface of T-cells. The T-cell receptor as afamily exhibits polymorphic binding specificity similar toimmunoglobulins in its diversity. The mature T-cell receptor iscomprised of alpha and beta chains each having a variable (V) andconstant (C) region. The similarities that the T-cell receptor has toimmunoglobulins in genetic organization and function shows that T-cellreceptor contains regions of conserved sequence. Lai et al., Nature,331:543-546 (1988).

Exemplary conserved sequences include those coding for amino acidresidues 84-90 of alpha chain, amino acid residues 107-115 of betachain, and amino acid residues 91-95 and 111-116 of the gamma chain.Kabat et al., supra, p. 279.

Integrins and Adhesions

Adhesive proteins involved in cell attachment are members of a largefamily of related proteins termed integrins. Integrins are heterodimerscomprised of a beta and an alpha subunit. Members of the integrin familyinclude the cell surface glycoproteins platelet receptor GpIIb-IIIa,vitronectin, receptor (VnR) fibronectin receptor (FnR) and the leukocyteadhesion receptors LFA-1, Mac-1, Mo-1 and 60.3. Roushahti et al.,Science, 238:491-497 (1987). Nucleic acid and protein sequence datademonstrates regions of conserved sequences exist in the members ofthese families particularly between the beta chain of GpIIb-IIIa VnR andFnR, and between the alpha subunit of VnR, Mac-1, LFA-1, Fnr andGpIIb-IIIa. Suzuki et al., Proc. Natl. Acad. Sci. USA, 83:8614-8618,1986; Ginsberg et al., J. Biol. Chem., 262:5437-5440, 1987.

The following discussion illustrates the method of the present inventionapplied to isolating a conserved receptor-coding gene from theimmunoglobulin gene repertoire. This discussion is not to be taken aslimiting, but rather as illustrating application of principles that canbe used to isolate a gene from any family of conserved genes coding forfunctionally related receptors.

Generally, the method combines the following elements:

1. Isolating nucleic acids containing a substantial portion of theimmunological repertoire.

2. Preparing polynucleotide primers for cloning polynucleotide segmentscontaining immunoglobulin V_(H) and/or V_(L) region genes.

3. Preparing a gene library containing a plurality of different V_(H)and V_(L) genes from the repertoire.

4. Expressing the V_(H) and/or V_(L) polypeptides in a suitable host,including prokaryotic and eukaryotic hosts, either separately or in thesame cell, and either on the same or different expression vectors.

5. Screening the expressed polypeptides for the preselected activity,and segregating a V_(H)- and/or V_(L)-coding gene identified by thescreening process.

A receptor produced by the present invention assumes a conformationhaving a binding site specific for as evidenced by its ability to becompetitively inhibited, a preselected or predetermined ligand such asan antigen, enzymatic substrate and the like. In one embodiment, areceptor of this invention is a ligand binding polypeptide that forms anantigen binding site which specifically binds to a preselected antigento form a complex having a sufficiently strong binding between theantigen and the binding site for the complex to be isolated. When thereceptor is an antigen binding polypeptide its affinity or avidity isgenerally greater than 10⁵- M⁻¹ more usually greater than 10⁶ andpreferably greater than 10⁸ M⁻¹.

In another embodiment, a receptor of the subject invention binds asubstrate and catalyzes the formation of a product from the substrate.While the topology of the ligand binding site of a catalytic receptor isprobably more important for its preselected activity than its affinity(association constant or pKa) for the substrate, the subject catalyticreceptors have an association constant for the preselected substrategenerally greater than 10³ M⁻¹, more usually greater than 10⁵ M⁻¹ or 10⁶M⁻¹ and preferably greater than 10⁷ M⁻¹.

Preferably the receptor produced by the subject invention isheterodimeric and is therefore normally comprised of two differentpolypeptide chains, which together assume a conformation having abinding affinity, or association constant for the preselected ligandthat is different, preferably higher, than the affinity or associationconstant of either of the polypeptides alone, i.e., as monomers. One orboth of the different polypeptide chains is derived from the variableregion of the light and heavy chains of an immunoglobulin. Typically,polypeptides comprising the light (V_(L)) and heavy (V_(H)) variableregions are employed together for binding the preselected ligand.

A receptor produced by the subject invention can be active in monomericas well as multimeric forms, either homomeric or heteromeric, preferablyheterodimeric. A V_(H) and V_(L) ligand binding polypeptide produced bythe present invention can be advantageously combined in the heterodimerto modulate the activity of either or to produce an activity unique tothe heterodimer. The individual ligand binding polypeptides will bereferred to as V_(H) and V_(L) and the heterodimer will be referred toas a Fv. However, it should be understood that a V_(H) bindingpolypeptide may contain in addition to the V_(H), substantially or aportion of the heavy chain constant region. A V_(L) binding polypeptidemay contain in addition to the V_(L), substantially all or a portion ofthe light chain constant region. A heterodimer comprised of a V_(H)binding polypeptide containing a portion of the heavy chain constantregion and a V_(L) binding containing substantially all of the lightchain constant region is termed a Fab fragment. The production of Fabcan be advantageous in some situations because the additional constantregion sequences contained in a Fab as compared to a F_(V) couldstabilize the V_(H) and V_(L) interaction. Such stabilization couldcause the Fab to have higher affinity for antigen. In addition the Fabis more commonly used in the art and thus there are more commercialantibodies available to specifically recognize a Fab.

The individual V_(H) and V_(L) polypeptides will generally have fewerthan 125 amino acid residues, more usually fewer than about 120 aminoacid residues, while normally having greater than 60 amino acidresidues, usually greater than about 95 amino acid residues, moreusually greater than about 100 amino acid residues. Preferably, theV_(H) will be from about 110 to about 125 amino acid residues in lengthwhile V_(L) will be from about 95 to about 115 amino acid residues inlength.

The amino acid residue sequences will vary widely, depending upon theparticular idiotype involved. Usually, there will be at least twocysteines separated by from about 60 to 75 amino acid residues andjoined by a disulfide bond. The polypeptides produced by the subjectinvention will normally be substantial copies of idotypes of thevariable regions of the heavy and/or light chains of immunoglobulins,but in some situations a polypeptide may contain random mutations inamino acid residue sequences in order to advantageously improve thedesired activity.

In some situations, it is desirable to provide for covalent crosslinking of the V_(H) and V_(L) polypeptides, which can be accomplishedby providing cysteine resides at the carboxyl termini. The polypeptidewill normally be prepared free of the immunoglobulin constant regions,however a small portion of the J region may be included as a result ofthe advantageous selection of DNA synthesis primers. The D region willnormally be included in the transcript of the V_(H).

In other situations, it is desirable to provide a peptide linker toconnect the V_(L) and the V_(H) to form a single-chain antigen-bindingprotein comprised of a V_(H) and a V_(L). This single-chainantigen-binding protein would be synthesized as a single protein chain.Such single-chain antigen-binding proteins have been described by Birdet al., Science, 242:423-426 (1988). The design of suitable peptidelinker regions is described in U.S. Pat. No. 4,704,692 by RobertLandner.

Such a peptide linker could be designed as part of the nucleic acidsequences contained in the expression vector. The nucleic acid sequencescoding for the peptide linker would be between the V_(H) and V_(L) DNAhomologs and the restriction endonuclease sites used to operatively linkthe V_(H) an V_(L) DNA homologs to the expression vector.

Such a peptide linker could also be coded for nucleic acid sequencesthat are part of the polynucleotide primers used to prepare the variousgene libraries. The nucleic acid sequence coding for the peptide linkercan be made up of nucleic acids attached to one of the primers or thenucleic acid sequence coding for the peptide linker may be derived fromnucleic acid sequences that are attached to several polynucleotideprimers used to create the gene libraries.

Typically the C terminus region of the V_(H) and V_(L) polypeptides willhave a greater variety of the sequences than the N terminus and, basedon the present strategy, can be further modified to permit a variationof the normally occurring V_(H) and V_(L) chains. A syntheticpolynucleotide can be employed to vary one or more amino in anhypervariable region.

1. Isolation of the Repertoire

To prepare a composition of nucleic acids containing a substantialportion of the immunological gene repertoire, a source of genes codingfor the V_(H) and/or V_(L) polypeptides is required. Preferably thesource will be a heterogeneous population of antibody producing cells,i.e. B lymphocytes (B cells), preferably rearranged B cells such asthose found in the circulation or spleen of a vertebrate. (Rearranged Bcells are those in which immunoglobulin gene translocation, i.e.,rearrangement, has occurred as evidenced by the presence in the cell ofmRNA with the immunoglobulin gene V, D and J region transcriptsadjacently located thereon.)

In some cases, it is desirable to bias the repertoire for a preselectedactivity, such as by using as a source of nucleic acid cells (sourcecells) from vertebrates in any one of various stages of age, health andimmune response. For example, repeated immunization of a healthy animalprior to collecting rearranged B cells results in obtaining a repertoireenriched for genetic material producing a ligand binding polypeptide ofhigh affinity. Conversely, collecting rearranged B cells from a healthyanimal whose immune system has not been recently challenged results inproducing a repertoire that is not biased towards the production of highaffinity V_(H) and/or V_(L) polypeptides.

It should be noted the greater the genetic heterogeneity of thepopulation of cells for which the nucleic acids are obtained, thegreater the diversity of the immunological repertoire that will be madeavailable for screening according to the method of the presentinvention. Thus, cells from different individuals, particularly thosehaving an immunologically significant age difference, and cells fromindividuals of different strains, races or species can be advantageouslycombined to increase the heterogeneity of the repertoire.

Thus, in one preferred embodiment, the source cells are obtained from avertebrate, preferably a mammal, which has been immunized or partiallyimmunized with an antigenic ligand (antigen) against which activity issought, i.e., a preselected antigen. The immunization can be carried outconventionally. Antibody titer in the animal can be monitored todetermine the stage of immunization desired, which stage corresponds tothe amount of enrichment or biasing of the repertoire desired. Partiallyimmunized animals typically receive only one immunization and cells arecollected therefrom shortly after a response is detected. Fullyimmunized animals display a peak titer, which is achieved with one ormore repeated injections of the antigen into the host mammal, normallyat 2 to 3 week intervals. Usually three to five days after the lastchallenge, the spleen is removed and the genetic repertoire of thespleenocytes, about 90% of which are rearranged B cells, is isolatedusing standard procedures. See, Current Protocols in Molecular Biology,Ausubel et al., eds., John Wiley & Sons, NY.

Nucleic acids coding for V_(H) and V_(L) polypeptides can be derivedfrom cells producing IgA, IgD, IgE, IgG or IgM, most preferably from IgMand IgG, producing cells.

Methods for preparing fragments of genomic DNA from which immunoglobulinvariable region genes can be cloned as a diverse population are wellknown in the art. See for example Herrmann et al., Methods In Enzymol.,152:180-183, (1987); Frischauf Methods In Enzymol., 152:183-190 (1987);Frischauf, Methods In Enzymol., 152:190-199 (1987); and DiLella et al.,Methods In Enzymol., 152:199-212 (1987). (The teachings of thereferences cited herein are hereby incorporated by reference.)

The desired gene repertoire can be isolated from either genomic materialcontaining the gene expressing the variable region or the messenger RNA(mRNA) which represents a transcript of the variable region. Thedifficulty in using the genomic DNA from other than non-rearranged Blymphocytes is in juxtaposing the sequences coding for the variableregion, where the sequences are separated by introns. The DNAfragment(s) containing the proper exons must be isolated, the intronsexcised, and the exons then spliced in the proper order and in theproper orientation. For the most part, this will be difficult, so thatthe alternative technique employing rearranged B cells will be themethod of choice because the C D and J immunoglobulin gene regions havetranslocated to become adjacent, so that the sequence is continuous(free of introns) for the entire variable regions.

Where mRNA is utilized the cells will be lysed under RNase inhibitingconditions. In one embodiment, the first step is to isolate the totalcellular mRNA by hybridization to an oligo-dT cellulose column. Thepresence of mRNAs coding for the heavy and/or light chain polypeptidescan then be assayed by hybridization with DNA single strands of theappropriate genes. Conveniently, the sequences coding for the constantportion of the V_(H) and V_(L) can be used as polynucleotide probes,which sequences can be obtained from available sources. See for example,Early and Hood, Genetic Engineering, Setlow and Hollaender, eds., Vol.3, Plenum Publishing Corporation, New York, (1981), pages 157-188; andKabat et al., Sequences of Immunological Interest, National Institutesof Health, Bethesda, Md., (1987).

In preferred embodiments, the preparation containing the total cellularmRNA is first enriched for the presence of V_(H) and/or V_(L) codingmRNA. Enrichment is typically accomplished by subjecting the total mRNApreparation or partially purified mRNA product thereof to a primerextension reaction employing a polynucleotide synthesis primer of thepresent invention.

2. Preparation of Polynucleotide Primers

The term “polynucleotide” as used herein in reference to primers, probesand nucleic acid fragments or segments to be synthesized by primerextension is defined as a molecule comprised of two or moredeoxyribonucleotides or ribonucleotides, preferably more than 3. Itsexact size will depend on many factors, which in turn depends on theultimate conditions of use.

The term “primer” as used herein refers to a polynucleotide whetherpurified from a nucleic acid restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product which is complimentary to a nucleic acid strand isinduced, i.e., in the presence of nucleotides and an agent forpolymerization such as DNA polymerase, reverse transcriptase and thelike, and at a suitable temperature and pH. The primer is preferablysingle stranded for maximum efficiency, but may alternatively be doublestranded. If double stranded, the primer is first treated to separateits strands before being used to prepare extension products. Preferably,the primer is a polydeoxyribonucleotide. The primer must be sufficientlylong to prime the synthesis of extension products in the presence of theagents for polymerization. The exact lengths of the primers will dependon may factors, including temperature and the source of primer. Forexample, depending on the complexity of the target sequence, apolynucleotide primer typically contains 15 to 25 or more nucleotides,although it can contain fewer nucleotides. Short primer moleculesgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with template.

The primers used herein are selected to be “substantially” complementaryto the different strands of each specific sequence to be synthesized oramplified. This means that the primer must be sufficiently complementaryto nonrandomly hybridize with its respective template strand. Therefore,the primer sequence may not reflect the exact sequence of the template.For example, a non-complementary nucleotide fragment can be attached tothe 5′ end of the primer, with the remainder of the primer sequencebeing substantially complementary to the strand. Such noncomplementaryfragments typically code for an endonuclease restriction site.Alternatively, noncomplementary bases or longer sequences can beinterspersed into the primer, provided the primer sequence hassufficient complementarily with the sequence of the strand to besynthesized or amplified to non-randomly hybridize therewith and therebyform an extension product under polynucleotide synthesizing conditions.

The polynucleotide primers can be prepared using any suitable method,such as, for example, the phosphotriester on phosphodiester methods seeNarang et al., Meth. Enzymol., 68:90, (1979); U.S. Pat. No. 4,356,270;and Brown et al., Meth. Enzymol., 68:109, (1979).

The choice of a primer's nucleotide sequence depends on factors such asthe distance on the nucleic acid from the region coding for the desiredreceptor, its hybridization site on the nucleic acid relative to anysecond primer to be used, the number of genes in the repertoire it is tohybridize to, and the like.

For example, to produce V_(H)-coding DNA homologs by primer extension,the nucleotide sequence of a primer is selected to hybridize with aplurality of immunoglobulin heavy chain genes at a site substantiallyadjacent to the V_(H)-coding region so that a nucleotide sequence codingfor a functional (capable of binding) polypeptide is obtained. Tohybridize to a plurality of different V_(H)-coding nucleic acid strands,the primer must be a substantial complement of a nucleotide sequenceconserved among the different strands. Such sites include nucleotidesequences in the constant region, any of the variable region frameworkregions, preferably the third framework region, leader region, promoterregion, J region and the like.

Primers of the present invention may also contain a DNA-dependent RNApolymerase promoter sequence or its complement. See for example, Krieget al., Nucleic Acids Research, 12:7057-70 (1984); Studier et al., J.Mol. Biol., 189:113-130 (1986); and Molecular Cloning: A LaboratoryManual, Second Edition, Maniatis et al., eds. Cold Spring Harbor, N.Y.(1989).

When a primer containing a DNA-dependent RNA polymerase promoter is usedthe primer is hybridized to the polynucleotide strand to be amplifiedand the second polynucleotide strand of the DNA-dependent RAN polymerasepromoter is completed using an inducing such as E. coli, DNA polymeraseI, or the Klenow fragment of E. Coli DNA polymerase I. The complementaryRNA polynucleotide is then produced by adding an RNA-dependent RNApolymerase. The starting polynucleotide is amplified by alternatingbetween the production of an RNA polynucleotide and DNA polynucleotide.

3. Preparing a Gene Library

The strategy used for cloning, i.e., substantially reproducing, theV_(H) and/or V_(L) genes contained within the isolated repertoire willdepend, as is well known in the art, on the type, complexity, and purityof the nucleic acids making up the repertoire. Other factors includewhether or not the genes are to be amplified and/or mutigenized.

In one strategy, the object is to clone the V_(H)- and/or V_(L)-codinggenes from a repertoire comprised of polynucleotide coding strands, suchas mRNA and/or the sense strand of genomic DNA. If the repertoire is inthe form of double stranded genomic DNA, it is usually first denatured,typically by melting, into single strands. The repertoire is subjectedto a first primary extension reaction by treating (contacting) therepertoire with a first polynucleotide synthesis primer having apreselected nucleotide sequence. The first primer is capable ofinitiating the first primer extension reaction by hybridizing to anucleotide sequence, preferably at least about 10 nucleotides in lengthand more preferably at least about 20 nucleotides in length, conservedwithin the repertoire. The first primer is sometimes referred to hereinas the “sense primer” because it hybridizes to the coding or sensestrand of a nucleic acid. In addition, the second primer is sometimesreferred to herein as the “anti-sense primer” because it hybridizes to anon-coding or anti-sense strand of a nucleic acid, i.e., a strandcomplementary to a coding strand.

The first primer extension is performed by mixing the first primer,preferably a predetermined amount thereof, with the nucleic acids of therepertoire, preferably a predetermined amount thereof, to form a firstprimer extension reaction admixture. The admixture is maintained underpolynucleotide synthesizing conditions for a time period, which istypically predetermined, sufficient for the formation of a first primerextension reaction product, thereby producing a plurality of differentV_(H)-coding DNA homolog complements. The complements are then subjectedto a second primer extension reaction by treating them with a secondpolynucleotide synthesis primer having a preselected nucleotidesequence. The second primer is capable of initiating the second reactionby hybridizing to a nucleotide sequence, preferably at least about 10nucleotides in length and more preferably at least about 20 nucleotidesin length, conserved among a plurality of different V_(H)-coding genecomplements such as those, for example, produced by the first primerextension reaction. This is accomplished by mixing the second primer,preferably a predetermined amount thereof, with the compliment nucleicacids, preferably a predetermined amount thereof, to form a secondprimer extension reaction admixture. The admixture is maintained underpolynucleotide synthesizing conditions for a time period, which istypically predetermined, sufficient for the formation of a first primerextension reaction product, thereby producing a gene library containinga plurality of different V_(H)-and/or V_(L)-coding DNA homologs.

In another strategy, the object is to clone the V_(H)- and/orV_(L)-coding genes from a repertoire by providing a polynucleotidecomplement of the repertoire, such as the anti-sense strand of genomicdsDNA or the polynucleotide produced by subjecting mRNA to a reversetranscriptase reaction. Methods for producing such complements are wellknown in the art. The complement is subjected to a primer extensionreaction similar to the above-described second primer extensionreaction, i.e., a primer extension reaction using a polynucleotidesynthesis primer capable of hybridizing to a nucleotide sequenceconserved among a plurality of different V_(H)-coding gene complements.

The primer extension reaction is performed using any suitable method.Generally it occurs in a buffered aqueous solution, preferably at a pHof 7-9, most preferably about 8. Preferably, a molar excess (for genomicnucleic acid, usually about 10⁶:1 primer:template) of the primer isadmixed to the buffer containing the template strand. A large molarexcess is preferred to improve the efficiency of the process.

The deoxyribonucleotide triphosphates DATP, dCTP, dGTP, and dTTP arealso admixed to the primer extension (polynucleotide synthesis) reactionadmixture in adequate amounts and the resulting solution is heated toabout 90 C.-100 C. for about 1 to 10 minutes, preferably from 1 to 4minutes. After this heating period the solution is allowed to cool toroom temperature, which is preferable for primer hybridization. To thecooled mixture is added an appropriate agent for inducing or catalyzingthe primer extension reaction, and the reaction is allowed to occurunder conditions known in the art. The synthesis reaction may occur atfrom room temperature up to a temperature above which the inducing agentno longer functions efficiently. Thus, for example, if DNA polymerase isused as inducing agent, the temperature is generally no greater thanabout 40 C.

The inducing agent may be any compound or system which will function toaccomplish the synthesis of primer extension products, includingenzymes. Suitable enzymes for this purpose include, for example, E.coli, DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4DNA polymerase, other available DNA polymerases, reverse transcriptase,and other enzymes, including heat-stable enzymes, which will facilitatecombination of the nucleotides in the proper manner to form the primerextension products which are complementary to each nucleic acid strand.Generally, the synthesis will be initiated at the 3′ end of each primerand proceed in the 5′ direction along the template strand, untilsynthesis terminates, producing molecules of different lengths. Theremay be inducing agents, however, which initiate synthesis at the 5′ endand proceed in the above direction, using the same process as describedabove.

The inducing agent also may be a compound or system which will functionto accomplish the synthesis of RNA primer extension products, includingenzymes. In preferred embodiments, the inducing agent may be aDNA-dependent RNA polymerase such as T7 RNA polymerase, T3 RNApolymerase or SP6 RNA polymerase. These RNA polymerases initiatesynthesis from a promoter contained within a primer of the presentinvention. These polymerases produce a complementary RNA polynucleotide.The high turn over rate of the RNA polymerase amplifies the startingpolynucleotide as has been described by Chamberlin et al., The Enzymes,ed., P. Boyer, P P. 87-108, Academic Press, New York (1982). Anotheradvantage of T7 RNA polymerase is that mutations can be introduced intothe polynucleotide synthesis by replacing a portion of cDNA with one ormore mutagenic oligodeoxynucleotides (polynucleotides) and transcribingthe partially-mismatched template directly as has been previouslydescribed by Joyce et al., Nucleic Acid Research, 17:711-722 (1989).

If the inducing agent is a DNA-dependent RNA polymerase and thereforeincorporates ribonucleotide triphosphates, sufficient amounts of ATP,CTP, GTP, and UTP are admixed to the primer extension reaction admixtureand the resulting solution is treated as described above.

The newly synthesized strand and its complementary nucleic acid strandform a double-stranded molecule which can be used in the succeedingsteps of the process.

The first and/or second primer extension reaction discussed above canadvantageously be used to incorporate into the receptor a preselectedepitope useful in immunologically detecting and/or isolating a receptor.This is accomplished by utilizing a first and/or second polynucleotidesynthesis primer or expression vector to incorporate a predeterminedamino acid residue sequence into the amino acid residue sequence of thereceptor.

After producing V_(H)- and/or V_(L)-coding DNA homologs for a pluralityof different V_(H)- and/or V_(L)-coding genes within the repertoire, thehomologs are typically amplified. While the V_(H) and/or V_(L)-codingDNA homologs can be amplified by classic techniques such asincorporation into an autonomously replicating vector, it is preferredto first amplify the DNA homologs by subjecting them to a polymerasechain reaction (PCR) prior to inserting them into a vector. In fact, inpreferred strategies, the first and/or second primer extension reactionsused to produce the gene library are the first and second primerextension reactions in a polymerase chain reaction.

PCR is carried out by cycling i.e., simultaneously performing in oneadmixture, the above described first and second primer extensionreactions, each cycle comprising polynucleotide synthesis followed bydenaturation of the double stranded polynucleotides formed. Methods andsystems for amplifying a DNA homolog are described in U.S. Pat. Nos.4,683,195 and 4,683,202, both to Mullis et al.

In preferred embodiments, the PCR process is used not only to amplifythe V_(H)- and/or V_(L)-coding DNA homologs of the library, but also toinduce mutations within the library and thereby provide a library havinga greater heterogeneity. First, it should be noted that the PCRprocesses itself is inherently mutagenic due to a variety of factorswell known in the art. Second, in addition to the mutation inducingvariations described in the above referenced U.S. Pat. No. 4,683,195,other mutation inducing PCR variations can be employed. For example, thePCR reaction admixture, i.e., the combined first and second primerextension reaction admixtures, can be formed with different amounts ofone or more of the nucleotides to be incorporated into the extensionproduct. Under such conditions, the PCR reaction proceeds to producenucleotide substitutions within the extension product as a result of thescarcity of a particular base. Similarly, approximately equal molaramounts of the nucleotides can be incorporated into the initial PCRreaction admixture in an amount to efficiently perform X number ofcycles, and then cycling the admixture through a number of cycles inexcess of X, such as, for instance, 2X. Alternatively, mutations can beinduced during the PCR reaction by incorporating into the reactionadmixture nucleotide derivatives such as inosine, not normally found inthe nucleic acids of the repertoire being amplified. During subsequentin vivo amplification, the nucleotide derivative will be replaced with asubstitute nucleotide thereby inducing a point mutation.

4. Expressing the V_(H) and/or V_(L) DNA Homologs

The V_(H)- and/or V_(L)-coding DNA homologs contained within the libraryproduced by the above-described method can be operatively linked to avector for amplification and/or expression.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. One type ofpreferred vector is an episome, i.e., a nucleic acid molecule capable ofextra-chromosomal replication. Preferred vectors are those capable ofautonomous replication and/or expression of nucleic acids to which theyare linked. Vectors capable of directing the expression of genes towhich they are operatively linked are referred to herein as “expressionvectors”.

The choice of vector to which a V_(H)- and/or V_(L)-coding DNA homologis operatively linked depends directly, as is well known in the art, onthe functional properties desired, e.g., replication or proteinexpression, and the host cell to be transformed, these being limitationsinherent in the art of constructing recombinant DNA molecules.

In preferred embodiments, the vector utilized includes a procaryoticreplicon i.e., a DNA sequence having the ability to direct autonomousreplication and maintenance of the recombinant DNA molecule extrachromosomally in a procaryotic host cell, such as a bacterial host cell,transformed therewith. Such replicons are well known in the art. Inaddition, those embodiments that include a procaryotic replicon alsoinclude a gene whose expression confers a selective advantage, such asdrug resistance, to a bacterial host transformed therewith. Typicalbacterial drug resistance genes are those that confer resistance toampicillin or tetracycline.

Those vectors that include a procaryotic replicon can also include aprocaryotic promoter capable of directing the expression (transcriptionand translation) of the V_(H)- and/or V_(L)-coding homologs in abacterial host cell, such as E. coli transformed therewith. A promoteris an expression control element formed by a DNA sequence that permitsbinding of RNA polymerase and transcription to occur. Promoter sequencescompatible with bacterial hosts are typically provided in plasmidvectors containing convenience restriction sites for insertion of a DNAsegment of the present invention. Typical of such vector plasmids arepUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratories,(Richmond, Calif.) and pPL and pKK223 available from Pharmacia,(Piscataway, N.J.).

Expression vectors compatible with eucaryotic cells, preferably thosecompatible with vertebrate cells, can also be used. Eucaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAhomologue. Typical of such vectors are pSVL and pKSV-10 (Pharmacia),pBPV-1/PML2d (International Biotechnologies, Inc.), and pTDT1 (ATCC, No.31255).

In preferred embodiments, the eucaryotic cell expression vectors usedinclude a selection marker that is effective in an eucaryotic cell,preferably a drug resistant selection marker. A preferred drugresistance marker is the gene whose expression results in neomycinresistance, i.e., the neomycin phosphotransferase (neo) gene. Southernet al., J. Mol. Appl. Genet., 1:327-341 (1982).

The use of retroviral expression vectors to express the genes of theV_(H) and/or V_(L)-coding DNA homologs is also contemplated. As usedherein, the term “retroviral expression vector” refers to a DNA moleculethat includes a promoter sequences derived from the long terminal repeat(LTR) region of a retrovirus genome.

In preferred embodiments, the expression vector is typically aretroviral expression vector that is preferably replication-incompetentin eucaryotic cells. The construction and use of retroviral vectors hasbeen described by Sorge et al., Mol. Cel. Biol., 4:1730-1737 (1984).

A variety of methods have been developed to operatively link DNA tovectors via complementary cohesive termini. For instance, complementarycohesive termini can be engineered into the V_(H)- and/or V_(L)-codingDNA homologs during the primer extension reaction by use of anappropriately designed polynucleotide synthesis primer, as previouslydiscussed. The vector, and DNA homolog if necessary, is cleaved with arestriction endonuclease to produce termini complementary to those ofthe DNA homolog. The complementary cohesive termini of the vector andthe DNA homolog are then operatively linked (ligated) to produce aunitary double stranded DNA molecule.

In preferred embodiments, the V_(H)-coding and V_(L)-coding DNA homologsof diverse libraries are randomly combined in vitro for polycistronicexpression from individual vectors. That is, a diverse population ofdouble stranded DNA expression vectors is produced wherein each vectorexpresses, under the control of a single promoter, one V_(H)-coding DNAhomolog and one V_(L)-coding DNA homolog, the diversity of thepopulation being the result of different V_(H)- and V_(L)-coding DNAhomolog combinations.

Random combination in vitro can be accomplished using two expressionvectors distinguished from one another by the location on each of arestriction site common to both. Preferably the vectors are lineardouble stranded DNA, such as a Lambda Zap derived vector as describedherein. In the first vector, the site is located between a promoter anda polylinker, i.e., 5′ terminal (upstream relative to the direction ofexpression) to the polylinker but 3′ terminal (downstream relative tothe direction of expression). In the second vector, the polylinker islocated between a promoter and the restriction site, i.e., therestriction site is located 3′ terminal to the polylinker, and thepolylinker is located 3′ terminal to the promoter.

In preferred embodiments, each of the vectors defines a nucleotidesequence coding for a ribosome binding and a leader, the sequence beinglocated between the promoter and the polylinker, but downstream (3′terminal) from the shared restriction site if that site is between thepromoter and polylinker. Also preferred are vectors containing a stopcodon downstream from the polylinker, but upstream from any sharedrestriction site if that site is downstream from the polylinker. Thefirst and/or second vector can also define a nucleotide sequence codingfor a peptide tag. The tag sequence is typically located downstream fromthe polylinker but upstream from any stop codon that may be present.

In preferred embodiments, the vectors contain selectable markers suchthat the presence of a portion of that vector, i.e. a particular lambdaarm, can be selected for or selected against. Typical selectable markersare well known to those skilled in the art. Examples of such markers areantibiotic resistance genes, genetically selectable markers, mutationsuppressors such as amber suppressors and the like. The selectablemarkers are typically located upstream of the promoter and/or downstreamof the second restriction site. In preferred embodiments, one selectablemarker is located upstream of the promoter on the first vectorcontaining the V_(H)-coding DNA homologs. A second selectable marker islocated downstream of the second restriction site on the vectorcontaining the V_(L)-coding DNA homologs. This second selectable markermay be the same or different from the first as long as when theV_(H)-coding vectors and the V_(L)-coding vectors are randomly combinedvia the first restriction site the resulting vectors containing bothV_(H) and V_(L) and both selectable markers can be selected.

Typically the polylinker is a nucleotide sequence that defines one ormore, preferably at least two, restriction sites, each unique to thevector and preferably not shared by the other vector, i.e., if it is onthe first vector, it is not on the second vector. The polylinkerrestriction sites are oriented to permit ligation of V_(H)- orV_(L)-coding DNA homologs into the vector in same reading frame as anyleader, tag or stop codon sequence present.

Random combination is accomplished by ligating V_(H)-coding DNA homologsinto the first vector, typically at a restriction site or sites withinthe polylinker. Similarly, V_(L)-coding DNA homologs are ligated intothe second vector, thereby creating two diverse populations ofexpression vectors. It does not matter which type of DNA homolog, i.e.,V_(H) or V_(L), is ligated to which vector, but it is preferred, forexample, that all V_(H)-coding DNA homologs are ligated to either thefirst or second vector, and all of the V_(L)-coding DNA homologs areligated to the other of the first or second vector. The members of bothpopulations are then cleaved with an endonuclease at the sharedrestriction site, typically by digesting both populations with the sameenzyme. The resulting product is two diverse populations of restrictionfragments where the members of one have cohesive termini complementaryto the cohesive termini of the members of the other. The restrictionfragments of the two populations are randomly ligated to one another,i.e., a random, interpopulation ligation is performed, to produce adiverse population of vectors each having a V_(H)-coding andV_(L)-coding DNA homolog located in the same reading frame and under thecontrol of second vector's promoter. Of course, subsequentrecombinations can be effected through cleavage at the sharedrestriction site, which is typically reformed upon ligation of membersfrom the two populations, followed by subsequent religations.

The resulting construct is then introduced into an appropriate host toprovide amplification and/or expression of the V_(H)- and/orV_(L)-coding DNA homologs, either separately or in combination. Whencoexpressed within the same organism, either on the same or thedifferent vectors, a functionally active Fv is produced. When the V_(H)and V_(L) polypeptides are expressed in different organisms, therespective polypeptides are isolated and then combined in an appropriatemedium to form a Fv. Cellular hosts into which a V_(H)- and/orV_(L)-coding DNA homolog-containing construct has been introduced arereferred to herein as having been “transformed” or as “transformants”.

The host cell can be either procaryotic or eucaryotic. Bacterial cellsare preferred procaryotic host cells and typically are a strain of E.coli such as, for example, the E. coli strain DH5 available fromBethesda Research Laboratories, Inc., Bethesda, Md. Preferred eucaryotichost cells include yeast and mammalian cells, preferably vertebratecells such as those from a mouse, rat, monkey or human cell line.

Transformation of appropriate cell hosts with a recombinant DNA moleculeof the present invention is accomplished by methods that typicallydepend on the type of vector used. With regard to transformation ofprocaryotic host cells, see, for example, Cohen et al., ProceedingsNational Academy of Science, USA, Vol. 69, P. 2110 (1972); and Maniatiset al., Molecular Cloning, a Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1982). With regard to thetransformation of vertebrate cells with retroviral vectors containingrDNAs, see for example, Sorge et al., Mol. Cell. Biol., 4:1730-1737(1984); Graham et al., Virol., 52:456 (1973); and Wigler et al.,Proceedings National Academy of Sciences, USA, Vol. 76, P. 1373-1376(1979).

5. Screening for Expression of V_(H) and/or V_(L) Polypeptides

Successfully transformed cells, i.e., cells containing a V_(H)- and/orV_(L)-coding DNA homolog operatively linked to a vector, can beidentified by any suitable well known technique for detecting thebinding of a receptor to a ligand or the presence of a polynucleotidecoding for the receptor, preferably its active site. Preferred screeningassays are those where the binding of ligand by the receptor produces adetectable signal, either directly or indirectly. Such signals include,for example, the production of a complex, formation of a catalyticreaction product, the release or uptake of energy, and the like. Forexample, cells from a population subjected to transformation with asubject rDNA can be cloned to produce monoclonal colonies. Cells formthose colonies can be harvested, lysed and their DNA content examinedfor the presence of the rDNA using a method such as that described bySouthern, J. Mol. Biol., 98:503 (1975) or Berent et al., Biotech. 3:208(1985).

In addition to directly assaying for the presence of a V_(H)- and/orV_(L)-coding DNA homolog, successful transformation can be confirmed bywell known immunological methods, especially when the V_(H) and/or V_(L)polypeptides produced contain a preselected epitope. For example,samples of cells suspected of being transformed are assayed for thepresence of the preselected epitope using an antibody against theepitope.

6. V_(H)- and/or V_(L)-Coding Gene Libraries

The present invention contemplates a gene library, preferably producedby a primer extension reaction or combination of primer extensionreactions as described herein, containing at least about 10³, preferablyat least about 10⁴ and more preferably at least about 10⁵ differentV_(H)- and/or V_(L)-coding DNA homologs. The homologs are preferably inan isolated form, that is, substantially free of materials such as, forexample, primer extension reaction agents and/or substrates, genomic DNAsegments, and the like.

In preferred embodiments, a substantial portion of the homologs presentin the library are operatively linked to a vector, preferablyoperatively linked for expression to an expression vector.

Preferably, the homologs are present in a medium suitable for in vitromanipulation, such as water, water containing buffering salts, and thelike. The medium should be compatible with maintaining the biologicalactivity of the homologs. In addition, the homologs should be present ata concentration sufficient to allow transformation of a host cellcompatible therewith at reasonable frequencies.

It is further preferred that the homologs be present in compatible hostcells transformed therewith.

D. Expression Vectors

The present invention also contemplates various expression vectorsuseful in performing, inter alia, the methods of the present invention.Each of the expression vectors is a novel derivative of Lambda Zap.

1. Lambda Zap II

Lambda Zap II is prepared by replacing the Lambda S gene of the vectorLambda Zap with the Lambda S gene from the Lambda gt10 vector, asdescribed in Example 6.

2. Lambda Zap II V_(H)

Lambda Zap II V_(H) is prepared by inserting the synthetic DNA sequencesillustrated in FIG. 6A into the above-described Lambda Zap II vector.The inserted nucleotide sequence advantageously provides a ribosomebinding site (Shine-Dalgarno sequence) to permit proper imitation ofmRNA translation into protein, and a leader sequence to efficientlydirect the translated protein to the periplasm. The preparation ofLambda Zap II V_(H) is described in more detail in Example 9, and itsfeatures illustrated in FIGS. 6A and 7.

3. Lambda Zap II V_(L)

Lambda Zap II V_(L) is prepared as described in Example 12 by insertinginto Lambda Zap II the synthetic DNa sequence illustrated in FIG. 6B.Important features of Lambda Zap II V_(L) are illustrated in FIG. 8.

4. Lambda Zap II V_(L) II

Lambda Zapp II V_(L) II is prepared as described in Example 11 byinserting into Lambda Zap II the synthetic DNA sequence illustrated inFIG. 10.

The above-described vectors are compatible with E. coli hosts, i.e.,they can express for secretion into the periplasm proteins coded for bygenes to which they have been operatively linked for expression.

EXAMPLES

The following examples are intended to illustrate, but not limit, thescope of the invention.

1. Polynucleotide Selection

The nucleotide sequences encoding the immunoglobulin protein CDR's arehighly variable. However, there are several regions of conservedsequences that flank the V_(H) domains. For instance, containsubstantially conserved nucleotide sequences, i.e., sequences that willhybridize to the same primer sequence. Therefore, polynucleotidesynthesis (amplification) primers that hybridize to the conservedsequences and incorporate restriction sites into the DNA homologproduced that are suitable for operatively linking the synthesized DNAfragments to a vector were constructed. More specifically, the DNAhomologs were inserted into Lambda ZAP II vector (Stratagene CloningSystem, San Diego, Calif.) at the Xho I and EcoR I sites. Foramplification of the V_(H) domains, the 3′ primer (primer 12 in Table1), was designed to be complementary to the mRNA in the J_(H) region. Inall cases, the 5′ primers (primers 1-10, Table 1) (SEQ ID NOS: 49-68)were chosen to be complementary to the first strand cDNA in theconserved N-terminus region (antisense strand). Initially amplificationwas performed with a mixture of 32 primers (primer 1, Table 1) that weredegenerate at five positions. Hybridoma mRNA could be amplified withmixed primers, but initial attempts to amplify mRNA from spleen yieldedvariable results. Therefore, several alternatives to amplification usingthe mixed 5′ primers were compared.

The first alternative was to construct multiple unique primers, eight ofwhich are shown in Table 1, corresponding to individual members of themixed primer pool. The individual primers 2-9 of Table 1 wereconstructed by incorporating either of the two possible nucleotides atthree of the five degenerate positions.

The second alternative was to construct a primer containing inosine(primer 10, Table 1) at four of the variable positions based on thepublished work of Takahashi, et al., Proc. Natl. Acad. Sci. (U.S.A.),82:1931-1935, (1985) and Ohtsuka et al., J. Biol. Chem., 260: 2605-2608,(1985). This primer has the advantage that it is not degenerate and, atthe same time minimizes the negative effects of mismatches at theunconserved positions as discussed by Martin et al., Nuc. Acids Res.,13:8927 (1985). However, it was not known if the presence of inosinenucleotides would result in incorporation of unwanted sequences in thecloned V_(H) regions. Therefore, inosine was not included at the oneposition that remains in the amplified fragments after the cleavage ofthe restriction sites. As a result, inosine was not in the clonedinsert.

Additional, V_(H) amplification primers including the unique 3′ primerwere designed to be complementary to a portion of the first constantregion domain of the gamma 1 heavy chain mRNA (primers 15 and 16, TableI). These primers will produce DNA homologs containing polynucleotidescoding for amino acids from the V_(H) and the first constant regiondomains of the heavy chain. These DNA homologs can therefore be used toproduce Fab fragments rather than an F_(V).

As a control for amplification from spleen or hybridoma mRNA, a set ofprimers hybridizing to a highly conserved region within the constantregion IgG, heavy chain gene were constructed. The 5′ primer (11,Table 1) is complementary to the cDNA in the C_(H)2 region whereas the3′ primer (13, Table 1) is complementary to the mRNA in the C_(H)3region. It is believed that no mismatches were present between theseprimers and their templates.

The nucleotide sequences encoding the V_(L) CDRs are highly variable.However, there are several regions of conserved sequences that flank theV_(L) CDR domains including the J_(L), V_(L) framework regions and V_(L)leader/promotor. Therefore, amplification primers that hybridize to theconserved sequences and incorporate restriction sites that allowingcloning the amplified fragments into the pBluescript SK-vector cut withNcoI and SpeI were constructed. For amplification of the V_(L) CDRdomains, the 3′ primer (primer number 14 in Table 1), was designed to becomplementary to the mRNA in the J_(L) regions. The 5′ primer (primer15, Table 1) was chosen to be complementary to the first strand cDNA inthe conserved N-terminus region (antisense strand).

A second set of amplification primers for amplification of the V_(L) CDRdomains the 5′ primers (primers 1-8 in Table II) (SEQ ID NOS: 69-79)were designed to be complementary to the first strand cDNA in theconserved N-terminus region. These primers also introduced a Sac Irestriction endonuclease site to allow the V_(L)DNA homolog to be clonedinto the V_(L)II-expression vector. The 3′ V_(L) amplification primer(primer 9 in Table II) was designed to be complementary to the mRNA inthe J_(L) regions and to introduce the Xba I restriction endonucleasesite required to insert the V_(L)DNA homolog into the V_(L)II-expressionvector (FIG. a).

Additional 3′ V_(L) amplification primers were designed to hybridize tothe constant region of either kappa or lambda mRNA (primers 10 and 11 inTable II). These primers allow a DNA homolog to be produced containingpolynucleotide sequences coding for constant region amino acids ofeither kappa or lambda chain. These primers make it possible to producean Fab fragment rather than an F_(V).

All primers and synthetic polynucleotides were either purchased fromResearch Genetics in Huntsville, Ala. or synthesized on an AppliedBiosystems DNA synthesizer, model 381A, using the manufacturer'sinstruction.

TABLE 1 (1) 5′AGGT(C/G)(C/A)A(G/A)CT(G/T)CTCGAGTC(T/A)GG 3′ degenerate5′ primer for the amplification of variable heavy chain region (V_(H))(2) 5′AGGTCCAGCTGCTCGAGTCTGG 3′ Unique 5′ primer for the amplificationof V_(H) (3) 5′AGGTCCAGCTGCTCGAGTCAGG 3′ Unique 5′ primer for theamplification of V_(H) (4) 5′AGGTCCAGCTTCTCGAGTCTGG 3′ Unique 5′ primerfor the amplification of V_(H) (5) G′AGGTCCAGCTTCTCGAGTCAGG 3′ Unique 5′primer for the amplification of V_(H) (6) 5′AGGTCCAACTGCTCGAGTCTGG 3′Unique 5′ primer for the amplification of V_(H) (7)5′AGGTCCAACTGCTCGAGTCAGG 3′ Unique 5′ primer for the amplification ofV_(H) (8) 5′AGGTCCAACTTCTCGAGTCTGG 3′ Unique 5′ primer for theamplification of V_(H) (9) 5′AGGTCCAACTTCTCGAGTCAGG 3′ Unique 5′ primerfor the amplification of V_(H) (10) 5′AGGTIIAICTICTCGAGTC(T/A) 3′ 5′degenerate primer containing inosine at 4 degenerate positions (11)5′GCCCAAGGATGTGCTCACC 3′ 5′ primer for amplification in the C_(H)2region of mouse IgG1 (12) 5′CTATTAGAATTCAACGGTAACAGTGGTGCCTTGGCCCCA 3′3′ primer for amplification of V_(H) (13) 5′CTCAGTATGGTGGTTGTGC 3′ 3′primer for amplification in the C_(H)3 region of mouse IgG1 (14)5′GCTACTAGTTTTGATTTCCACCTTGG 3′ 3′ primer for amplification of V_(L)(15) 5′CAGCCATGGCCGACATCCAGATG 3′ 5′ primer for amplification of V_(L)(16) 5′AATTTTACTAGTCACCTTGGTGCTGCTGGC 3′ Unique 3′ primer foramplification of V_(H) including part of the mouse gamma 1 firstconstant (17) 5′TATGCAACTAGTACAACCACAATCCCTGGGCACAATTTT 3′ Unique 3′primer for amplification of V_(H) including part of mouse gamma 1 firstconstant region and hinge region

TABLE II (1) 5′ CC AGT TCC GAG CTC GTT GTG ACT CAG GAA TCT 3′ Unique 5′primer for the amplification of V_(L) (2) 5′ CC AGT TCC GAG CTC GTG TTGACG CAG CCG CCC 3′ Unique 5′ primer for the amplification of V_(L) (3)5′ CC AGT TCC GAG CTC GTG CTC ACC CAG TCT CCA 3′ Unique 5′ primer forthe amplification of V_(L) (4) 5′ CC AGT TCC GAG CTC CAG ATG ACC CAG TCTCCA 3′ Unique 5′ primer for the amplification of V_(L) (5) 5′ CC AGA TGTGAG CTC GTG ATG ACC CAG ACT CCA 3′ Unique 5′ primer for theamplification of V_(L) (6) 5′ CC AGA TGT GAG CTC GTC ATG ACC CAG TCT CCA3′ Unique 5′ primer for the amplification of V_(L) (7) 5′ CC AGA TGT GAGCTC TTG ATG ACC CAA ACT CAA 3′ Unique 5′ primer for the amplification ofV_(L) (8) 5′ CC AGA TGT GAG CTC GTG ATA ACC CAG GAT GAA 3′ Unique 5′primer for the amplification of V_(L) (9) 5′ GC AGC ATT CTA GAG TTT CAGCTC CAG CTT GCC 3′ Unique 3′ primer for V_(L) amplification (10) 5′CCGCCGTCTAGAACACTCATTCCTGTTGAAGCT 3′ Unique 3′ primer for V_(L)amplification including CK (11) 5′ CCGCCGTCTAGAACATTCTGCAGGAGACAGACT 3′Unique 3′ primer for V_(L) amplification including C lambda

2. Production of a V_(H) Coding Repertoire Enriched in FITC BindingProteins

Fluorescein isothiocyanate (FITC) was selected as a ligand for receptorbinding. It was further decided to enrich by immunization theimmunological gene repertoire, i.e., V_(H)- and V_(l)-coding generepertoires, for genes coding for anti-FITC receptors. This wasaccomplished by linking FITC to keyhole limpet hemocyanin (KLH) usingthe techniques described in Antibodies a Laboratory Manual, Harlow andLowe, eds., Cold Spring Harbor, N.Y., (1988). Briefly, 10.0 milligrams(mg) of keyhole limpet hemocyanin and 0.5 mg of FITC were added to 1 mlof buffer containing 0.1 M sodium carbonate at pH 9.6. and stirred for18 to 24 hours at 4 degrees C. (4 C.). The unbound FITC was removed bygel filtration through Sephadex G-25.

The KLH-FITC conjugate was prepared for injection into mice by adding100 μg of the conjugate to 250 μl of phosphate buffered saline (PBS). Anequal volume of complete Freund's adjuvant was added and emulsified theentire solution for 5 minutes. A 129 G_(IX+) mouse was injected with 300μl of the emulsion. Injections were given subcutaneously at severalsites using a 21 gauge needle. A second immunization with KLH-FITC wasgiven two weeks later. This injection was prepared as follows: fifty μgof KLH-FITC were diluted in 250 μL of PBS and an equal volume of alumwas admixed to the KLH-FITC solution. The mouse was injectedintraperitoneally with 500 μl of the solution using a 23 gauge needle.One month later the mice were given a final injection of 50 μg of theKLH-FITC conjugate diluted to 200 μL in PBS. This injection was givenintravenously in the lateral tail vein using a 30 gauge needle. Fivedays after this final injection the mice were sacrificed and totalcellular RNA was isolated from their spleens.

Hybridoma PCP 8D11 producing an antibody immunospecific for phosphonateester was cultured in DMEM media (Gibco Laboratories, Grand Island,N.Y.) containing 10 percent fetal calf serum supplemented withpenicillin and streptomycin. About 5×10⁸ hybridoma cells were harvestedand washed twice in phosphate buffered saline. Total cellular RNA wasprepared from these isolated hybridoma cells.

3. Preparation of a V_(H)-Coding Gene Repertoire

Total cellular RNA was prepared from the spleen of a single mouseimmunized with KLH-FITC as described in Example 2 using the RNApreparation methods described by Chomczynski et al., Anal Biochem.,162:156-159 (1987)using the manufacturer's instructions and the RNAisolation kit produced by Stratagene Cloning Systems, La Jolla, Calif.Briefly, immediately after removing the spleen from the immunized mouse,the tissue was homogenized in 10 ml of a denaturing solution containing4.0 M guanide isothiocyanate, 0.25 M sodium citrate at pH 7.0, and 0.1 M2-mercaptoethanol using a glass homogenizer. One ml of sodium acetate ata concentration of 2 M at pH 4.0 was admixed with the homogenizedspleen. One ml of phenol that had been previously saturated with H₂O wasalso admixed to the denaturing solution containing the homogenizedspleen. Two ml of a chloroform:isoamyl alcohol (24:1 v/v) mixture wasadded to this homogenate. The homogenate was mixed vigorously for tenseconds and maintained on ice for minutes. The homogenate was thentransferred to a thick-walled 50 ml polypropylene centrifuged tube(Fisher Scientific Company, Pittsburg, Pa.). The solution wascentrifuged at 10,000×g for 20 minutes at 4 C. The upper RNA-containingaqueous layer was transferred to a fresh 50 ml polypropylene centrifugetube and mixed with an equal volume of isopropyl alcohol. This solutionwas maintained at −20 C. for at least one hour to precipitate the RNA.The solution containing the precipitated RNA was centrifuged at 10,000×gfor twenty minutes at 4 C. The pelleted total cellular RNA was collectedand dissolved in 3 ml of the denaturing solution described above. Threeml of isopropyl alcohol was added to the resuspended total cellular RNAand vigorously mixed. This solution was maintained at −20 C. for atleast 1 hour to precipitate the RNA. The solution containing theprecipitated RNA was centrifuged at 10,000×g for ten minutes at 4 C. Thepelleted RNA was washed once with a solution containing 75% ethanol. Thepelleted RNA was dried under vacuum for 15 minutes and then resuspendedin dimethyl pyrocarbonate (DEPC) treated (DEPC-H₂O) H₂O.

Messenger RNA (mRNA) enriched for sequences containing long poly Atracts was prepared from the total cellular RNA using methods describedin Molecular Cloning A Laboratory Manual, Maniatias et al., eds., ColdSpring Harbor Laboratory, New York, (1982). Briefly, one half of thetotal RNA isolated from a single immunized mouse spleen prepared asdescribed above was resuspended in one ml of DEPC-H₂O and maintained at65 C. for five minutes. One ml of 2× high salt loading buffer consistingof 100 mM Tris-HCL, 1 M sodium chloride, 2.0 mM disodium ethylenediamine tetraacetic acid (EDTA) at pH 7.5, and 0.2% sodium dodecylsulfate (SDS) was added to the resuspended RNA and the mixture allowedto cool to room temperature. The mixture was then applied to an oligo-dT(Collaborative Research Type 2 or Type 3) column that was previouslyprepared by washing the oligo-dT with a solution containing 0.1 M sodiumhydroxide and 5 mM EDTA and then equilibrating the column with DEPC-H₂O.The eluate was collected in a sterile polypropylene tube and reappliedto the same column after heating the eluate for 5 minutes at 65 C. Theoligo dT column was then washed with 2 ml of high salt loading bufferconsisting of 50 mM Tris-HCL at pH 7.5, 500 mM sodium chloride, 1 mMEDTA at pH 7.5 and 0.1% SDS. The oligo dT column was then washed with 2ml of 1× medium salt buffer consisting of 50 mM Tris-HCL at pH 7.5, 100mM sodium chloride 1 mM EDTA and 0.1% SDS. The messenger RNA was elutedfrom the oligo dT column with 1 ml of buffer consisting of 10 mMTris-HCL at pH 7.5, 1 mM EDTA at pH 7.5 and 0.05% SDS. The messenger RNAwas purified by extracting this solution with phenol/chloroform followedby a single extraction with 100% chloroform. The messenger RNA wasconcentrated by ethanol precipitation and resuspended in DEPC H₂O.

The messenger RNA isolated by the above process contains a plurality ofdifferent V_(H) coding polynucleotides, i.e., greater than about 10⁴different V_(H)-coding genes.

4. Preparation of a Single V_(H) Coding Polynucleotide

Polynucleotides coding for a single V_(H) were isolated according toExample 3 except total cellular RNA was extracted from monoclonalhybridoma cells prepared in Example 2. The polynucleotides isolated inthis manner code for a single V_(H).

5. DNA Homolog Preparation

In preparation for PCR amplification, mRNA prepared according to theabove examples was used as a template for cDNA synthesis by a primerextension reaction. In a typical 50 ul transcription reaction, 5-10 ugof spleen or hybridoma mRNA in water was first hybridized (annealed)with 500 ng (50.0 pmol) of the 3′ V_(H) primer (12, Table 1), at 65 C.for five minutes. Subsequently, the mixture was adjusted to 1.5 mM DATP,dCTP, dGTP and dTTP, 40 mM Tris-HCl at pH 8.0, 8 mM MgCl₂, 50 mM NaCl,and 2 mM spermidine. Moloney-Murine Leukemia virus Reverse transcriptase(Stratagene Cloning Systems), 26 units, was added and the solution wasmaintained for 1 hour at 37 C.

PCR amplification was performed in a 100 ul reaction containing theproducts of the reverse transcription reaction (approximately 5 ug ofthe cDNA/RNA hybrid), 300 ng of 3′ V_(H) primer (primer 12 of Table 1),300 ng each of the 5′ V_(H) primers (primer 2-10 of Table 1) 200 mM of amixture of dNTP's, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 15 mM MgCl₂, 0.1%gelatin and 2 units of Taq DNA polymerase. The reaction mixture wasoverlaid with mineral oil and subjected to 40 cycles of amplification.Each amplification cycle involved denaturation at 92 C. for 1 minute,annealing at 52 C. for 2 minutes and polynucleotide synthesis by Primerextension (elongation) at 72 C. for 1.5 minutes. The amplifiedV_(H)-coding DNA homolog containing samples were extracted twice withphenol/chloroform, once with chloroform, ethanol precipitated and- werestored at −70 C. in 10 mM Tris-HCl, (pH, 7.5) and 1 mM EDTA.

Using unique 5′ primers (2-9, Table 1), efficient V_(H)-coding DNAhomolog synthesis and amplification from the spleen mRNA was achieved asshown in FIG. 3, lanes R17-R24. The amplified cDNA (V_(H)-coding DNAhomolog) is seen as a major band of the expected size (360 bp). Theintensities of the amplified V_(H)-coding polynucleotide fragment ineach reaction appear to be similar, indicating that all of these primersare about equally efficient in initiating amplification. The yield andquality of the amplification with these primers was reproducible.

The primer containing inosine also synthesized amplified V_(H)-codingDNA honologs from spleen mRNA reproducibly, leading to the production ofthe expected sized fragment, of an intensity similar to that of theother amplified cDNAs (FIG. 4, lane R16). This result indicated that thepresence of inosine also permits efficient DNA homolog synthesis andamplification. Clearly indicating how useful such primers are ingenerating a plurality of V_(H)-coding DNa homologs. Amplificationproducts obtained from the constant region primers (primers 11 and 13,Table 1) were more intense indicating that amplification was moreefficient, possibly because of a higher degree of homology between thetemplate and primers (FIG. 4, Lane R9). Based on these results, aV_(H)-coding gene library was constructed from the products of eightamplifications, each performed with a different 5′ primer. Equalportions of the products from each primer extension reaction were mixedand the mixed product was then used to generate a library ofV_(H)-coding DNA homolog-containing vectors.

DNA homologs of the V_(L) were prepared from the purified mRNA preparedas described above. In preparation for PCR amplification, mRNA preparedaccording to the above examples was used as a template for cDNAsynthesis. In a typical 50 ul transcription reaction, 5-10 ug of spleenor hybridoma mRNA in water was first annealed with 300 ng (50.0 pmol) ofthe 3′ V_(L) primer (14, Table 1), at 65 C. for five minutes.Subsequently,the mixture was adjusted to 1.5 mM DATP, dCTP, dGTP, anddTTP, 40 mM Tris-HCL at pH 8.0, 8 mM MgCl₂, 50 mM NaCl, and 2 mMspermidine. Moloney-Murine Leukemia virus reverse transcriptase(Stratagene Cloning Systems), 26 units, was added and the solution wasmaintained for 1 hour at 37 C. The PCR amplification was performed in a100 ul reaction containing approximately 5 ug of the cDNA/RNA hybridproduced as described above, 300 ng of the 3′ V_(L) primer (primer 14 ofTable 1), 300 ng of the 5′ V_(L) primer (primer 15 of Table 1), 200 mMof a mixture of dNTP's, 50 mM KCl, 10 mM Tris-HCl pH 8.3, 15 mM MgCl₂,0.1% gelatin and 2 units of Tag DNA polymerase. The reaction mixture wasoverlaid with mineral oil and subjected to 40 cycles of amplification.Each amplification cycle involved denaturation at 92 C. for 1 minute,annealing at 52 C. for 2 minutes and elongation at 72 C. for 1.5minutes. The amplified samples were extracted twice withphenol/chloroform, once with chloroform, ethanol precipitated and werestored at −70 C. in 10 mM Tris-HCl at 7.5 and 1 mM EDTA.

6. Inserting DNA Homologs into Vectors

In preparation for cloning a library enriched in V_(H) sequences, PCRamplified products (2.5 mg/30 ul of 150 mM NaCl, 8 mM Tris-HCl (pH 7.5),6 mM MgSO₄, 1 mM DTT, 200 mg/ml bovine serum albumin (BSA) at 37 C. weredigested with restriction enzymes Xho I (125 units) and EcoR I (10 U)and purified on a 1% agarose gel. In cloning experiments which requireda mixture of the products of the amplification reactions, equal volumes(50 ul, 1-10 ug concentration) of each reaction mixture were combinedafter amplification but before restriction digestion. After gelelectrophoresis of the digested PCR amplified spleen mRNA, the region ofthe gel containing DNA fragments of approximately 350 bps was excised,electroeluted into a dialysis membrane, ethanol precipitated andresuspended in 10 mM Tris-HCl pH 7.5 and 1 mM EDTA to a finalconcentration of 10 ng/ul. Equimolar amounts of the insert were thenligated overnight at 5 C. to 1 ug of Lambda ZAP™ II vector (StratageneCloning Systems, La Jolla, Calif.) previously cut by EcoR I and Xho I. Aportion of the ligation mixture (1 ul) was packaged for 2 hours at roomtemperature using Gigapack Gold packaging extract (Stratagene CloningSystems, La Jolla, Calif.), and the packaged material was plated onXL1-blue host cells. The library was determined to consist of 2×10⁷V_(H) homologs with less than 30% non-recombinant background.

The vector used above, Lambda Zap II is a derivative of the originalLambda Zap (ATCC #40,298) that maintains all of the characteristics ofthe original Lambda Zap including 6 unique cloning sites, fusion proteinexpression, and the ability to rapidly excise the insert-in the form ofa phagemid (Bluescript SK−), but lacks the SAM 100 mutation, allowinggrowth on many Non-Sup F strains, including XL1-Blue. The Lambda Zap IIwas constructed as described in Short et al., Nucleic Acids Res.,16:7583-7600, 1988, by replacing the Lambda S gene contained in a 4254base pair (bp) DNA fragment produced by digesting Lambda Zap with therestriction enzyme NcoI. This 4254 bp DNA fragment was replaced with the4254 bp DNA fragment containing the Lambda S gene isolated from Lambdagt10 (ATCC #40,179) after digesting the vector with the restrictionenzyme NcoI. The 4254 bp DNA fragment isolated from lambda gt10 wasligated into the original Lambda Zap vector using T4 DNA ligase andstandard protocols for such procedures described in Current Protocols inMolecular Biology, Ausubel et al., eds., John Wiley and Sons, New York,1987.

In preparation of cloning a library enriched in V_(L) sequences, 2 ug ofPCR amplified products (2.5 mg/30 ul of 150 mM NaCl, 8 mM Tris-HCL (pH7.5), 6 mM Mg SO₄, 1 mM DTT, 200 mg/ml BSA. 37 C.) were digested withrestriction enzymes Nco I (30 units) and Spe I (45 units). The digestedPCR amplified products were purified on a 1% agarose gel using standardelectroelution technique described in Molecular Cloning A LaboratoryManual, Maniatis et al., eds., Cold Spring Harbor, N.Y., (1982).Briefly, after gel electroelution of the digested PCR amplified productthe region of the gel containing the V_(L)-coding DNA fragment of theappropriate size was excised, electroelution into a dialysis membrane,ethanol precipitated and resuspended at a final concentration of 10 ngper ml in a solution containing 10 mM Tris-HCL at pH 7.5 and 1 mM EDTA.

An equal molar amount of DNA representing a plurality of differentV_(L)-coding DNA homologs was ligated to a pBluescript SK− phagemidvector that had been previously cut with Nco I and Spe I. A portion ofthe ligation mixture was transformed using the manufacturer'sinstructions into Epicuian Coli XL1-Blue competent cells (StragageneCloning Systems, La Jolla, Calif.). The transformant library wasdetermined to consist of 1.2×10³ colony forming units/ug of V_(L)homologs with less than 3% non-recombinant background.

7. Sequencing of Plasmids from the V_(H)-Coding cDNA Library

To analyze the Lambda Zap II phage clones the clones were excised fromLambda Zap into plasmids according to the manufacture's instructions(Stratagene Cloning System, La Jolla, Calif.). Briefly, phage plaqueswere cored from the agar plates and transferred to sterile microfugetubes containing 500 μl a buffer containing 50 mM Tris-HCL at pH 7.5,100 mM NaCl, 10 mM MgSO₄, and 0.01% gelatin and 20 uL of chloroform.

For excisions, 200 ul of the phage stock, 200 ul of XL1-Blue cells(A₆₀₀=1.00) and 1 ul of R408 helper phage (1×10¹¹ pfu/ml) were incubatedat 37 C. for 15 minutes. The excised plasmids were infected intoXL1-Blue cells and plated onto LB plates containing ampicillin. Doublestranded DNA was prepared from the phagemid containing cells accordingto the methods described by Holmes et al., Anal. Biochem., 114:193,(1981). Clones were first screened for DNA inserts by restrictiondigests with either Pvu II or Bg1 I and clones containing the putativeV_(H) insert were sequenced using reverse transcriptase according to thegeneral method described by Sanger et al., Proc. Natl. Acad. Sci., USA,74:5463-5467, (1977) and the specific modifications of this methodprovided in the manufacturer's instructions in the AMV reversetranscriptase ³⁵S-dATP sequencing kit from Stratagene Cloning Systems,La Jolla, Calif.

8. Characterization of the Cloned V_(H) Repertoire

The amplified products which had been digested with Xho I and EcoR I andcloned into Lambda ZAP, resulted in a cDNA library with 9.0×10⁵ pfu's.In order to confirm that the library consisted of a diverse populationof V_(H)-coding DNA homologs, the N-terminal 120 bases of 18 clones,selected at random from the library, were excised and sequenced (FIG.5). To determine if the clones were of V_(H) gene origin, the clonedsequences were compared with known V_(H) sequences and V_(L) sequences.The clones exhibited from 80 to 90% homology with sequences of knownheavy chain origin and little homology with sequences of light chainorigin when compared with the sequences available in Sequences ofProteins of Immunological Interest by Kabot et al., 4th ed., U.S.Dept.of Health and Human Sciences, (1987). This demonstrated that thelibrary was enriched for the desired V_(H) sequence in preference toother sequences, such as light chain sequences.

The diversity of the population was assessed by classifying thesequenced clones into predefined subgroups (FIG. 5). Mouse V_(H)sequences are classified into eleven subgroups (FIG. 5). Mouse V_(H)sequences are classified into eleven subgroups [I (A,B,), II (A,B,C),III (A,B,C,D,) V (A,B)] based on framework amino acid sequencesdescribed in Sequences of Proteins of Immunological Interest by Kabot etal., 4th ed., U.S. Dept. of Health and Human Sciences, (1987); Dildrop,Immunology Today, 5:84, (1984); and Brodeur et al., Eur. J. Immunol.,14; 922, (1984). Classification of the sequenced clones demonstratedthat the cDNA library contained V_(H) sequences of at least 7 differentsubgroups. Further, a pairwise comparison of the homology between thesequenced clones showed that no two sequences were identical at allpositions, suggesting that the population is diverse to the extent thatit is possible to characterize by sequence analysis.

Six of the clones (L 36-50, FIG. 5) belong to the subclass III B and hadvery similar nucleotide sequences. This may reflect a preponderance ofmRNA derived from one or several related variable genes in stimulatedspleen, but the data does not permit ruling out the possibility of abias in the amplification process.

9. V_(H)-Expression Vector Construction

To express the plurality of V_(H)-coding DNA homologs in an E. coli hostcell, a vector was constructed that placed the V_(H)-coding DNA homologsin the proper reading frame, provided a ribosome binding site asdescribed by Shine et al., Nature, 254:34, 1975, provided a leadersequence directing the expressed protein to the periplasmic space,provided a polynucleotide sequence that coded for a known epitope(epitope tag) and also provided a polynucleotide that coded for a spacerprotein between the V_(H)-coding DNA homolog and the polynucleotidecoding for the epitope tag. A synthetic DNA sequence containing all ofthe above polynucleotides and features was constructed by designingsingle stranded polynucleotide segments of 20-40 bases that wouldhybridize to each other and form the double stranded synthetic DNAsequence shown in FIG. 6. The individual single-stranded polynucleotides(N₁-N₁₂) are shown in Table III.

Polynucleotides 2, 3, 9-4′, 11, 10-5′, 6, 7 and 8 were kinased by adding1 μl of each polynucleotide (0.1 ug/ul) and 20 units of T₄polynucleotide kinase to a solution containing 70 mM Tris-HCL at pH 7.6,10 mM MgCl₂, 5 mM DTT, 10 mM 2ME, 500 micrograms per ml of BSA. Thesolution was maintained at 37 C. for 30 minutes and the reaction stoppedby maintaining the solution at 65 C. for 10 minutes. The two endpolynucleotides 20 ng of polynucleotides N1 and polynucleotides N12,were added to the above kinasing reaction solution together with 1/10volume of a solution containing 20.0 mM Tris-HCL at pH 7.4, 2.0 mM MgCl₂and 50.0 mM NaCl. This solution was heated to 70 C. for 5 minutes andallowed to cool to room temperature, approximately 25 C., over 1.5 hoursin a 500 ml beaker of water. During this time period all polynucleotidesannealed to form the double stranded synthetic DNA insert shown in FIG.6A. The individual polynucleotides were covalently linked to each otherto stabilize the synthetic DNA insert by adding 40 μl of the abovereaction to a solution containing 50 mM Tris-HCL at pH 7.5, 7 mM MgCl₂,1 mM DTT, 1 mM adenosine triphosphate (ATP) and 10 units of T4 DNAligase. This solution was maintained at 37 C. for 30 minutes and thenthe T4 DNA ligase was inactivated by maintaining the solution at 65 C.for 10 minutes. The end polynucleotides were kinased by mixing 52 μl ofthe above reaction, 4 μl of a solution containing 10 mM ATP and 5 unitsof T4 polynucleotide kinase. This solution was maintained at 37 C. for30 minutes and then the T4 polynucleotide kinase was inactivated bymaintaining the solution at 65 C. for 10 minutes. The completedsynthetic DNA insert was ligated directly into a lambda Zap II vectorthat had been previously digested with the restriction enzymes NotI andXhoI. The ligation mixture was packaged according to the manufacture'sinstructions using Gigapack II Gold packing extract available fromStratagene Cloning Systems, La Jolla, Calif. The packaged ligationmixture was plated on XL1blue cells (Stratagene Cloning Systems, SanDiego, Calif.). Individual lambda Zap II plaques were cored and theinserts excised according to the in vivo excision protocol provided bythe manufacturer, Stratagene Cloning Systems, La Jolla, Calif. This invivo excision protocol moves the cloned insert from the lambda Zap IIvector into a plasmid vector to allow easy manipulation and sequencing.The accuracy of the above cloning steps was confirmed by sequencing theinsert using the Sanger dideoxide method described in by Sanger et al.,Proc. Natl. Acad. Sci USA, 74:5463-5467, (1977) and using themanufacture's instructions in the AMV Reverse Transcriptase ³⁵S-ATPsequencing kit from Stratagene Cloning Systems, La Jolla, Calif. Thesequence of the resulting V_(H) expression vector is shown in FIG. 6Aand FIG. 7.

TABLE III N1) 5′ GGCCGCAAATTCTATTTCAAGGAGACAGTCAT 3′ N2)5′ AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3′ N3)5′ GTTATTACTCGCTGCCCAACCAGCCATGGCCC 3′ N4)5′ AGGTGAAACTGCTCGAGAATTCTAGACTAGGTTAATAG 3′ N5)5′ TCGACTATTAACTAGTCTAGAATTCTCGAG 3′ N6)5′ CAGTTTCACCTGGGCCATGGCTGGTTGGG 3′ N7)5′ CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG 3′ N8)5′ GTATTTCATTATGACTGTCTCCTTGAAATAGAATTTGC 3′ N9-4)5′ AGGTGAAACTGCTCGAGATTTCTAGACTAGTTACCCGTAC 3′ N11)5′ GACGTTCCGGACTACGGTTCTTAATAGAATTCG 3′ N12)5′ TCGACGAATTCTATTAAGAACCGTAGTC 3′ N10-5)5′ CGGAACGTCGTACGGGTAACTAGTCTAGAAATCTCGAG 3′

10. V_(L) Expression Vector Construction

To express the plurality of V_(L) coding polynucleotides in an E. colihost cell, a vector was constructed that placed the V_(L) codingpolynucleotide in the proper reading frame, provided a ribosome bindingsite as described by Shine et al., Nature, 254:34, (1975), provided aleader sequence directing the expressed protein to the periplasmic spaceand also provided a polynucleotide that coded for a spacer proteinbetween the V_(L) polynucleotide and the polynucleotide coding for theepitope tag. A synthetic DNA sequence containing all of the abovepolynucleotides and features was constructed by designing singlestranded polynucleotide segments of 20-40 bases that would hybridize toeach other and form the double stranded synthetic DNA sequence shown inFIG. 6B. The individual single-stranded polynucleotides (N₁-N₈) areshown in Table III.

Polynucleotides N2, N3, N4, N6, N7 and N8 were kinased by adding 1 μl ofeach polynucleotide and 20 units of T₄ polynucleotide kinase to asolution containing 70 mM Tris-HCL at pH 7.6, 10 mM MgCl₂, 5 mM DDT, 10mM 2ME, 500 micrograms per ml of BSA. The solution was maintained at 37C. for 30 minutes and the reaction stopped by maintaining the solutionat 65 C. for 10 minutes. The two end polynucleotides 20 ng ofpolynucleotides N1 and polynucleotides N5 were added to the abovekinasing reaction solution together with 1/10 volume of a solutioncontaining 20.0 mM Tris-HCL at pH 7.4, 2.0 mM MgCl₂ and 50.0 mM NaCl.This solution was heated to 70 C. for 5 minutes and allowed to cool toroom temperature, approximately 25 C., over 1.5 hours in a 500 ml beakerof water. During this time period all the polynucleotides annealed toform the double stranded synthetic DNA insert. The individualpolynucleotides were covalently linked to each other to stabilize thesynthetic DNA insert with adding 40 μl of the above reaction to asolution containing 50 ul Tris-HCL at pH 7.5, 7 mM MgCl₂, 1 mM DTT, 1 mMATP and 10 units of T4 DNA ligase. This solution was maintained at 37 C.for 30 minutes and then the T4 DNA ligase was inactivated by maintainingthe solution at 65 C. for 10 minutes. The end polynucleotides werekinased by mixing 52 μl of the above reaction, 4 μl of a solutionrecontaining 10 mM ATP and 5 units of T4 polynucleotide kinase. Thissolution was maintained at 37 C. for 30 minutes and then the T4polynucleotide kinase was inactivated by maintaining the solution at 65C. for 10 minutes. The completed synthetic DNA insert was ligateddirectly into a lambda Zap II vector that had been previously digestedwith the restriction enzymes NotI and XhoI. The ligation mixture waspackaged according to the manufacture's instructions using Gigapack IIGold packing extract available from Stratagene Cloning Systems, LaJolla, Calif. The packaged ligation mixture was plated on XL1-Blue cells(Stratagene Cloning Systems, La Jolla, Calif.). Individual lambda Zap IIplaques were cored and the inserts excised according to the in vivoexcision protocol provided by the manufacturer, Stratagene CloningSystems, La Jolla, Calif. and described in Short et al., Nucleic AcidsRes., 16:7583-7600, 1988. This in vivo excision protocol moves thecloned-insert from the lambda Zap II vector into a phagemid vector toallow easy manipulation and sequencing and also produces the phagemidversion of the V_(L) expression vectors. The accuracy of the abovecloning steps was confirmed by sequencing the insert using the Sangerdideoxide method described by Sanger et al., Proc. Natl. Acad. Aci. USA,74:5463-5467, (1977) and using the manufacturer's instructions in theAMV reverse transcriptase ³⁵S-DATP sequencing kit from StratageneCloning Systems, La Jolla, Calif. The sequence of the resulting V_(L)expression vector is shown in FIG. 6 and FIG. 8.

The V_(L) expression vector used to construct the V_(L) library was thephagemid produced to allow the DNA of the V_(L) expression vector to bedetermined. The phagemid was produced, as detailed above, by the in vivoexcision process from the Lambda Zap V_(L) expression vector (FIG. 8).The phagemid version of this vector was used because the Nco Irestriction enzyme site is unique in this version and thus could be usedto operatively linked the V_(L) DNA homologs into the expression vector.

11. V_(L)II-Expression Vector Construction

To express the plurality of V_(L)-coding DNA homologs in an E. coli hostcell, a vector was constructed that placed the V_(L)-coding DNA homologsin the proper reading frame, provided a ribosome binding site asdescribed by Shine et al., Nature, 254:34, 1975, provided the Pel B geneleader sequence that has been previously used to successfully secreteFab fragments in E. coli by Lei et al., J. Bac., 169:4379 (1987) andBetter et al., Science, 240:1041 (1988), and also provided apolynucleotide containing a restriction endonuclease site for cloning. Asynthetic DNA sequence containing all of the above polynucleotides andfeatures was constructed by designing single stranded polynucleotidesegments of 20-60 bases that would hybridize to each other and form thedouble stranded synthetic DNA sequence shown in FIG. 10. The sequence ofeach individual single-stranded polynucleotides (O₁-O₈) within thedouble stranded synthetic DNA sequence is shown in Table IV.

Polynucleotides 02, 03, 04, 05, 06 and 07 were kinased by adding 1 μl(0.1 ug/μl) of each polynucleotide and 20 units of T₄ polynucleotidekinase to a solution containing 70 mM Tris-HCL at pH 7.6, 10 mMmagnesium chloride (MgCl), 5 mM dithiothreitol (DTT), 10 mM2-mercaptoethanol (2ME), 500 micrograms per ml of bovine serum albumin.The solution was maintained at 37 C. for 30 minutes and the reactionstopped by maintaining the solution at 65 C. for 10 minutes. The 20 ngeach of the two end polynucleotides, 01 and 08, were added to the abovekinasing reaction solution together with 1/10 volume of a solutioncontaining 20.0 mM Tris-HCL at pH 7.4, 2.0 mM MgCl and 15.0 mM sodiumchloride (NaCl). This solution was heated to 70 C. for 5 minutes andallowed to cool to room temperature, approximately 25 C., over 1.5 hoursin a 500 ml beaker of water. During this time period all 8polynucleotides annealed to form the double stranded synthetic DNAinsert shown in FIG. 9. The individual polynucleotides were covalentlylinked to each other to stabilize the synthetic DNA insert by adding 40μl of the above reaction to a solution containing 50 ml Tris-HCL at pH7.5, 7 ml MgCl, 1 mm DTT, 1 mm ATP and 10 units of T4 DNA ligase. Thissolution was maintained at 37 C. for 30 minutes and then the T4 DNAligase was inactivated by maintaining the solution at 65 C. for 10minutes. The end polynucleotides were kinased by mixing 52 μl of theabove reaction, 4 μl of a solution containing 10 mM ATP and 5 units ofT4 polynucleotide kinase. This solution was maintained at 37 C. for 30minutes and then the T4 polynucleotide kinase was inactivated bymaintaining the solution at 65 C. for 10 minutes. The completedsynthetic DNA insert was ligated directly into a lambda Zap II vectorthat had been previously digested with the restriction enzymes Not I andXho I. The ligation mixture was packaged according to the manufacture'sinstructions using Gigapack II Gold packing extract available fromStratagene Cloning Systems, La Jolla, Calif. The packaged ligationmixture was plated on XL1blue cells (Stratagene Cloning Systems, SanDiego, Calif.). Individual lambda Zap II plaques were cored and theinserts excised according to the in vivo excision protocol provided bythe manufacturer, Stratagene Cloning Systems, La Jolla, Calif. This invivo excision protocol moves the cloned insert from the lambda Zap IIvector into a plasmid vector to allow easy manipulation and sequencing.The accuracy of the above cloning steps was confirmed by sequencing theinsert using the manufacture's instructions in the AMV ReverseTranscriptase ³⁵S-dATP sequencing kit from Stratagene Cloning Systems,La Jolla, Calif. The sequence of the resulting V_(L)II-expression vectoris shown in FIG. 9 and FIG. 11.

TABLE IV 01) 5′TGAATTCTAAACTAGTCGCCAAGGAGACAGTCAT 3′ 02)5′ AATGAAATACCTATTGCCTACGGCAGCCGCTGGATT 3′ 03)5′ GTTATTACTCGCTGCCCAACCAGCCATGGCC 3′ 04)5′ GAGCTCGTCAGTTCTAGAGTTAAGCGGCCG 3′ 05)5′ GTATTTCATTATGACTGTCTCCTTGGCGACTAGTTTAGAA-    TTCAAGCT 3′ 06)5′ CAGCGAGTAATAACAATCCAGCGGCTGCCGTAGGCAATAG 3′ 07)5′ TGACGAGCTCGGCCATGGCTGGTTGGG 3′ 08) 5′ TCGACGGCCGCTTAACTCTAGAAC 3′

12. V_(H)+V_(L) Library Construction

To prepare an expression library enriched in V_(H) sequences, DNAhomologs enriched in V_(H) sequences were prepared according to Example6 using the same set of 5′ primers but with primer 12A as the 3′ primer.These homologs were then digested with the restriction enzymes Xho I andSpe I and purified on a 1% agarose gel using the standard electroelutiontechnique described in Molecular Cloning A Laboratory Manual, Maniatiset al., eds., Cold Spring Harbor, N.Y., (1982). These prepared V_(H) DNAhomologs were then directly inserted into the V_(H) expression vectorthat had been previously digested with Xho I and Spe I.

The ligation mixture containing the V_(H) DNA homologs were packagedaccording to the manufacturers specifications using Gigapack Gold IIPacking Extract (Stratagene Cloning Systems, La Jolla, Calif.). Theexpression libraries were then ready to be plated on XL-1 Blue cells.

To prepare a library enriched in V_(L) sequences, PCR amplified productsenriched in V_(L) sequences were prepared according to Example 6. TheseV_(L) DNA homologs were digested with restriction enzymes Nco I and SpeI. The digested V_(L) DNA homologs were purified on a 1% agarose gelusing standard electroelusion techniques described in Molecular CloningA Laboratory Manual, Maniatis et al., eds., Cold Spring Harbor, N.Y.(1982). The prepared V_(L) DNA homologs were directly inserted into theV_(L) expression vector that had been previously digested with therestriction enzymes Nco I and Spe I. The ligation mixture containing theV_(L) DNA homologs were transformed into XL-1 blue competent cells usingthe manufacturer's instructions (Stratagene Cloning Systems, La Jolla,Calif.).

13. Inserting V_(L) Coding DNA Homologs into V_(L)II Expression Vector

In preparation for cloning a library enriched in V_(L) sequences, PCRamplified products (2.5 ug/30 ul of 150 mM NaCl, 8 mM Tris-HCl (pH 7.5),6 mM MgSO₄, 1 mM DTT, 200 ug/ml BSA at 37 C. were digested withrestriction enzymes Sac I (125 units) and Xba I (125 units) and purifiedon a 1% agarose gel. In cloning experiments which required a mixture ofthe products of the amplification reactions, equal volumes (50 ul, 1-10ug concentration) of each reaction mixture were combined afteramplification but before restriction digestion. After gelelectrophoresis of the digested PCR amplified spleen mRNA, the region ofthe gel containing DNA fragments of approximate 350 bps was excised,electroeluted into a dialysis membrane, ethanol precipitated andresuspended in a TE solution containing 10 mM Tris-HCl pH 7.5 and 1 mMEDTA to a final concentration of 50 ng/ul.

The V_(L)II-expression DNA vector was prepared for cloning by admixing100 ug of this DNA to a solution containing 250 units each of therestriction endonucleases Sac 1 and Xba 1 (both from BoehringerMannheim, Indianapolis, Ind.) and a buffer recommended by themanufacturer. This solution was maintained at 37 from 1.5 hours. Thesolution was heated at 65 C. for 15 minutes top inactivate therestriction endonucleases. The solution was chilled to 30 C. and 25units of heat-killable (HK) phosphatase (Epicenter, Madison, Wis.) andCaCl₂ were admixed to it according to the manufacturer's specifications.This solution was maintained at 30 C. for 1 hour. The DNA was purifiedby extracting the solution with a mixture of phenol and chloroformfollowed by ethanol precipitation. The V_(L)II expression vector was nowready for ligation to the V_(L) DNA homologs prepared in the aboveexamples.

DNA homologs enriched in V_(L) sequences were prepared according toExample 5 but using a 5′ light chain primer and the 3′ light chainprimer shown in Table II. Individual amplification reactions werecarried out using each 5′ light chain primer in combination with the 3′light chain primer. These separate V_(L) homolog containing reactionmixtures were mixed and digested with the restriction endonucleases Sac1 and Xba 1 according to Example 6. The V_(L) homologs were purified ona 1% agarose gel using the standard electroelution technique describedin Molecular Cloning A Laboratory Manual, Maniatis et al., eds., ColdSpring Harbor, N.Y., (1982). These prepared V_(L) DNA homologs were thendirectly inserted into the Sac 1-Xba cleaved V_(L)II-expression vectorthat was prepared above by ligating 3 moles of V_(L) DNA homolog insertswith each mole of the V_(L)II-expression vector overnight at 5 C.3.0×10⁵ plague forming units were obtained after packaging the DNA withGigapack II Bold (Stratagene Cloning Systems, La Jolla, Calif.) and 50%were recombinants.

14. Randomly Combining V_(H) and V_(L) DNA Homologs on the SameExpression Vector

The V_(L)II-expression library prepared in Example 13 was amplified and500 ug of V_(L)II-expression library phage DNA prepared from theamplified phage stock using the procedures described in MolecularCloning: A Laboratory Manual, Maniatis et al., eds., Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1982), 50 ug of thisV_(L)II-expression library phage DNA was maintained in a solutioncontaining 100 units of MLuI restriction endonuclease (BoehringerMannheim, Indianapolis, Ind.) in 200 ul of a buffer supplied by theendonuclease manufacturer for 1.5 hours at 37 C. The solution was thenextracted with a mixture of phenol and chloroform. The DNA was thenethanol precipitated and resuspended in 100 ul of water. This solutionwas admixed with 100 units of the restriction endonuclease EcoR I(Boehringer Mannheim, Indianapolis, Ind.) in a final volume of 200 ul ofbuffer containing the components specified by the manufacturer. Thissolution was maintained at 37 C. for 1.5 hours and the solution was thenextracted with a mixture of phenol and chloroform. The DNA was ethanolprecipitated and the DNA resuspended in TE.

The V_(H) expression library prepared in Example 12 was amplified and500 ug of V_(H) expression library phage DNA prepared using the methodsdetailed above. 50 ug of the V_(H) expression library phage DNA wasmaintained in a solution containing 100 units of Hind III restrictionendonuclease (Boehringer Mannheim, Indianapolis, Ind.) in 200 ul of abuffer supplied by the endonuclease manufacturer for 1.5 hours at 37 C.The solution was then extracted with a mixture of phenol and chloroformsaturated with 0.1 M Tris-HCL at pH 7.5. The DNA was then ethanolprecipitated and resuspended in 100 ul of water. This solution wasadmixed with 100 units of the restriction endonuclease EcoR I(Boehringer Mannheim, Indianapolis, Ind.) in a final volume of 200 ul ofbuffer containing the components specified by the manufacturer. Thissolution was maintained at 37 C. for 1.5 hours and the solution was thenextracted with a mixture of phenol and chloroform. The DNA was ethanolprecipitated and the DNA resuspended in TE.

The restriction digested V_(H) and V_(L)II-expression Libraries wereligated together. The ligation reaction consisted of 1 ug of V_(H) and 1ug of V_(L)II phage library DNA in a 10 ul reaction using the reagentssupplied in a ligation kit purchased from Stratagene Cloning Systems (LaJolla, Calif.). After ligation for 16 hr at 4 C., 1 ul of the ligatedthe phage DNA was packaged with Gigapack Gold II packaging extract andplated on XL 1-blue cells prepared according to the manufacturersinstructions. A portion of the 3×10⁶ clones obtained were used todetermine the effectiveness of the combination. The resulting V_(H) andV_(L) expression vector is shown in FIG. 11.

Clones containing both V_(H) and V_(L) were excised from the phage topBluescript using the in vitro excision protocol described by Short etal., Nucleic Acid Research, 16:7583-7600 (1988). Clones chosen forexcision expressed the decapetide tag and did not cleave X-gal in thepresence of 2 mM IPTGthus remaining white. Clones with thesecharacteristics represented 30% of the library. 50% of the clones chosenfor excision contained a V_(H) and V_(L) as determined by restrictionanalysis. Since approximately 30% of the clones in the V_(H) libraryexpressed the decapetide tag and 50% of the clones in the V_(L)IIlibrary contained a V_(L) sequence it was anticipated that no more than15% of the clones in the combined library would contain both V_(H) andV_(L) clones. The actual number obtained was 15% of the libraryindicating that the process of combination was very efficient.

15. Segregating DNA Homologs for a V_(H) Antigen Binding Protein

To segregate the individual clones containing DNA homologs that code fora V_(H) antigen binding protein, the title of the V_(H) expressionlibrary prepared according to Example 11 was determined. This librarytitration was performed using methods well known to one skilled in theart. Briefly, serial dilutions of the library were made into a buffercontaining 100 mM NaCl, 50 mM Tris-HCL at pH 7.5 and 10 MM MgSO₄. Ten ulof each dilution was added to 200 ul of exponentially growing E. colicells and maintained at 37 C. for 15 minutes to allow the phage toabsorb to the bacterial cells. Three ml of top agar consisting of 5 g/LNaCl, 2 g/L of MgSO₄, 5 g/L yeast extract, 10 g/L NZ amine (caseinhydrolysate) and 0.7% melted, 50 C. agarose. The phage, the bacteria andthe top agar were mixed and then evenly distributed across the surfaceof a prewarmed bacterial agar plate (5 g/L NaCl, 2 g/L MgSO₄, 5 g/Lyeast extract, 10 g/L NZ amine (casein hydrolysate) and 15 g/L Difcoagar. The plates were maintained at 37 C. for 12 to 24 hours duringwhich time period the lambda plaques developed on the bacterial lawn.The lambda plaques were counted to determined the total number of plaqueforming units per ml in the original library.

The titred expression library was then plated out so that replicafilters could be made from the library. The replica filters will be usedto later segregate out the individual clones in the library that areexpressing the antigens binding proteins of interest. Briefly, a volumeof the titred library that would yield 20,000 plaques per 150 millimeterplate was added to 600 ul of exponentially growing E. coli cells andmaintained at 37 C. for 15 minutes to allow the phage to absorb to thebacterial cells. Then 7.5 ml of top agar was admixed to the solutioncontaining the bacterial cells and the absorbed phage and the entiremixture distributed evenly across the surface of a prewarmed bacterialagar plate. This process was repeated for a sufficient number of platesto plate out a total number of plaques at least equal to the librarysize. These plates were then maintained at 37 C. for 5 hours. The plateswere then overlaid with nitrocellulose filters that had been pretreatedwith a solution containing 10 mM isopropyl-beta-D-thiogalactopyranosid(IPTG) and maintained at 37 C. for 4 hours. The orientation of thenitrocellulose filters in relation to the plate were marked by punchinga hole with a needle dipped in waterproof ink through the filter andinto the bacterial plates at several locations. The nitrocellulosefilters were removed with forceps and washed once in a TBST solutioncontaining 20 mM Tris-HCL at pH 7.5, 150 mM NaCl and 0.05% monolaurate(tween-20). A second nitrocellulose filter that had also been soaked ina solution containing 10 mM IPTG was reapplied to the bacterial platesto produce duplicate filters. The filters were further washed in a freshsolution of TBST for 15 minutes. Filters were then placed in a blockingsolution consisting of 20 mM Tris-HCL at pH 7.5, 150 mM NaCL and 1% BSAand agitated for 1 hour at room temperature. The nitrocellulose filterswere transferred to a fresh blocking solution containing a 1 to 500dilution of the primary antibody and gently agitated for at least 1 hourat room temperature. After the filters were agitated in the solutioncontaining the primary antibody the filters were washed 3 to 5 times inTBST for 5 minutes each time to remove any of the residual unboundprimary antibody. The filters were transferred into a solutioncontaining fresh blocking solution and a 1 to 500 to a 1 to 1,000dilution of alkaline phosphatase conjugated secondary antibody. Thefilters were gently agitated in the solution for at least 1 hour at roomtemperature. The filters were washed 3 to 5 times in a solution of TBSTfor at least 5 minutes each time to remove any residual unboundsecondary antibody. The filters were washed once in a solutioncontaining 20 mM Tris-HCL at pH 7.5 and 150 mM NaCL. The filters wereremoved from this solution and the excess moisture blotted from themwith filter paper. The color was developed by placing the filter in asolution containing 100 mM Tris-HCL at pH 9.5, 100 mM NaCl, 5 mM MgCl₂,0.3 mg/ml of nitro Blue Tetrazolium (NBT) and 0.15 mg/ml of5-bromo-4-chloro-3-indolyl-phosphate (BCIP) for at least 30 minutes atroom temperature. The residual color development solution was rinsedfrom the filter with a solution containing 20 mM Tris-HCL at pH 7.5 and150 mM NaCl. The filter was then placed in a stop solution consisting of20 mM Tris-HCL at pH 2.9 and 1 mM EDTA. The development of an intensepurple color indicates at positive result. The filters are used tolocate the phage plaque that produced the desired protein. That phageplaque is segregated and then grown up for further analysis.

Several different combinations of primary antibodies and secondantibodies were used. The first combination used a primary antibodyimmunospecific for a decapeptide that will be expressed only if theV_(H) antigen binding protein is expressed in the proper reading frameto allow read through translation to include the decapeptide epitopecovalently attached to the V_(H) antigen binding protein. Thisdecapeptide epitope and an antibody immunospecific for this decapeptideepitope was described by Green et al., Cell 28:477 (1982) and Niman etal., Proc. Nat. Acad. Sci. U.S.A. 80:4949 (1983). The sequence of thedecapeptide recognized is shown in FIG. 2. A functional equivalent ofthe monoclonal antibody that is immunospecific for the decapeptide canbe prepared according to the methods of Green et al. and Niman et al.The secondary antibody used with this primary antibody was a goatantimouse IgG (Fisher Scientific). This antibody was immunospecific forthe constant region of mouse IgG and did not recognize any portion ofthe variable region of heavy chain. This particular combination ofprimary and secondary antibodies when used according to the aboveprotocol determined that between 25% and 30% of the clones wereexpressing the decapeptide and therefore these clones were assumed toalso be expressing a V_(H) antigen binding protein.

In another combination the anti-decapeptide mouse monoclonal was used asthe primary antibody and an affinity purified goat anti-mouse Ig,commercially available as part of the picoBlue immunoscreening kit fromStratagene Cloning System; La Jolla, Calif., was use as the secondaryantibody. This combination resulted in a large number of false positiveclones because the secondary antibody also immunoreacted with the V_(H)of the heavy chain Therefore this antibody reacted with all clonesexpressing any V_(H) protein and this combination of primary andsecondary antibodies did not specifically detect clones with the V_(H)polynucleotide in the proper reading frame and thus allowing expressingof the decapeptide.

Several combinations of primary and secondary antibodies are used wherethe primary antibody is conjugated to fluorescein isothiobyanate (FITC)and thus the immunospecificity of the antibody was not important becausethe antibody is conjugated to the preselected antigen (FITC) and it isthat antigen that should be bound by the V_(H) antigen binding proteinsproduced by the clones in the expression library. After this primaryantibody has bound by virtue that is FITC conjugated mouse monoclonalantibody p2 5764 (ATCC #HB-9505). The secondary antibody used with thisprimary antibody is a goat anti-mouse Ig⁶ (Fisher Scientific, Pittsburg,Pa.) conjugated to alkaline phosphatase. Using the method described inAntibodies A Laboratory Manual, Harlow and Lowe, eds., Cold SpringingHarbor, New York, (1988). If a particular clone in the V_(H) expression,library, expresses a V_(H) binding protein that binds the FITCcovalently coupled to the primary antibody, the secondary antibody bindsspecifically and when developed the alkaline phosphate causes a distinctpurple color to form.

The second combination of antibodies of the type uses a primary antibodythat is FITC conjugated rabbit anti-human IgG (Fisher Scientific,Pittsburg, Pa.). The secondary antibody used with this primary antibodyis a goat anti-rabbit IgG conjugated to alkaline phosphatase using themethods described in Antibodies A Laboratory Manual, Harlow and Lane,eds., Cold Spring Harbor, N.Y., (1988). If a particular clone in theV_(H) expression library, expresses a V_(H) binding protein that bindsthe FITC conjugated to the primary antibody, the secondary antibodybinds specifically and when developed the alkaline phosphatase causes adistinct purple color to form.

Another primary antibody was the mouse monoclonal antibody (p2 5764(ATCC # HB-9505) conjugated to both FITC and ₁₂₅I. The antibody would bebound by any V_(H) antigen binding proteins expressed. Then because theantibody is also labeled with ¹²⁵I, an autoradiogram of the filter ismade instead of using a secondary antibody that is conjugated toalkaline phosphatase. This direct production of an autoradiogram allowssegregation of the clones in the library expressing a V_(H) antigenbinding protein of interest.

16. Segregating DNA Homologs for a V_(H) and V_(L) that Form an AntigenBinding F_(v)

To segregate the individual clones containing DNA homologs that code fora V_(H) and a V_(L) that form an antigen binding F_(V) the V_(H) andV_(L) expression library was titred according to Example 15. The titredexpression library was then screened for the presence of the decapetidetag expressed with the V_(H) using the methods described in Example 15.DNA was then prepared from the clones to express the decapepide tag.This DNA was digested with the restriction endonuclease Pvu II todetermine whether these clones also contained a V_(L) DNA homolog. Theslower migration of a PvuII restriction endonuclease fragment indicatedthat the particular clone contained both a V_(H) and a V_(L) DNAhomolog.

The clones containing both a V_(H) and a V_(L) DNA homolog were analyzedto determine whether these clones produced an assembled F_(V) proteinmolecule from the V_(H) and V_(L) DNA homologs.

The F_(V) protein fragment produced in clones containing both V_(H) andV_(L) was visualized by immune precipitation of radiolabled proteinexpressed in the clones. A 50 ml culture of LB broth (5 g/L yeastextract, 10 g/L and tryptone 10 g/L NaCl at pH 7.0) containing 100 ug/ulof ampicillin was inoculated with E. Coli harboring a plasmid contain aV_(H) and a V_(L). The culture was maintained at 37 C. with shakinguntil the optical density measured at 550 nm was 0.5 culture then wascentrifuged at 3,000 g for 10 minutes and resuspended in 50 ml of M9media (6 g/L Na₂HPO₄, 3 g/L KH₂PO₄, 0.5 g/L NaCl, 1 g/L NH₄Cl, 2g/Lglucose, 2 mM MgSO₄ and 0.1 mMgSO₄ CaCl₂ supplemented with amino acidswithout methionine or cysteine. This solution was maintained at 37 C.for 5 minutes and then 0.5 mCi of ³⁵S as HSO₄ ⁻ (New England Nuclear,Boston, Mass.) was added and the solution was further maintained at 37C. for an additional 2 hours. The solution was then centrifuged at3000×g and the supernatant discarded. The resulting bacterial cellpellet was frozen and thawed and then resuspended in a solutioncontaining 40 mM Tris pH 8.0, 100 mM sucrose and 1 mM EDTA. The solutionwas centrifuged at 10000×g for 10 minutes and the resulting pellettdiscarded. The supernatant was admixed with 10 ul of anti-decapeptidemonoclonal antibody and maintained for 30-90 minutes at on ice. 40 ul ofprotein G coupled to sepherose beads (Pharmacia, Piscataway, N.J.) wasadmixed to the solution and the added solution maintained for 30 minuteson ice to allow an immune precipitate to form. The solution wascentrifuged at 10,000×g for 10 minutes and the resulting pellet wasresuspended in 1 ml of a solution containing 100 mM Tris-HCL at pH 7.5and centrifuged at 10,000×g for 10 minutes. This procedure was repeatedtwice. The resulting immune precipitate pellet was loaded onto aPhastGel Homogenous 20 gel (Pharmacia, Piscataway, N.J.) according tothe manufacturer's directions. The gel was dried and used to exposeX-ray film.

The resulting autoradiogram is shown in FIG. 12. The presence ofassembled F_(V) molecules can be seen by the presence of V_(L) that wasimmunoprecipitated because it was attached to the V_(H)-decapepide tagrecognized by the precipitating antibody.

The foregoing is intended as illustrative of the present invention butnot limiting. Numerous variations and modifications can be effectedwithout departing from the true spirit and scope of the invention.

79 1 123 PRT Mouse VARIANT (1)...(123) HPCM2-hybridoma 1 Glu Val Lys LeuVal Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu ArgLeu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met GluTrp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala SerArg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val LysGly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu TyrLeu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr CysAla Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val 100 105 110 TrpGly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 2 123 PRT Mouse VARIANT(1)...(123) HPCM3-hybridoma 2 Glu Val Lys Leu Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr SerGly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro ProGly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn AspTyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val SerArg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala LeuArg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr Tyr GlySer Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr ValThr Val Ser Ser 115 120 3 123 PRT Mouse VARIANT (1)...(123)HPCM1-hybridoma 3 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val GlnPro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe ThrPhe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys ArgLeu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr ThrGlu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp ThrSer Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala GluAsp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser TyrTrp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr Val Thr Val SerSer 115 120 4 123 PRT Mouse VARIANT (1)...(123) HPCM6-hybridoma 4 GluVal Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 7580 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 9095 Tyr Cys Ala Arg Asp Tyr Tyr Asp Tyr Pro His Trp Tyr Phe Asp Val 100105 110 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 5 123 PRTMouse VARIANT (1)...(123) HPCM4-hybridoma 5 Glu Val Lys Leu Val Glu SerGly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser CysAla Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val ArgGln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn LysAla Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg PheIle Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln MetAsn Ala Leu Arg Ala Glu Asp Thr Ala Ile Phe 85 90 95 Tyr Cys Ala Arg AspTyr Tyr Arg Tyr Asp Gly Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala GlyThr Thr Val Thr Val Ser Ser 115 120 6 123 PRT Mouse 6 Glu Val Lys LeuVal Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu ArgLeu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met GluTrp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala SerArg Asn Lys Phe Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val LysGly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu TyrLeu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr CysAla Arg Asp Tyr Tyr Gly Ser Arg Tyr Trp Tyr Phe Asp Val 100 105 110 TrpGly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 7 123 PRT Mouse VARIANT(1)...(123) HPCG13-hybridoma 7 Glu Val Lys Leu Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Leu SerGly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Thr ProGly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Val Tyr Asn AspTyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val SerArg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala LeuArg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Ala Tyr GlySer Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr ValThr Val Ser Ser 115 120 8 123 PRT Mouse VARIANT (1)...(123)HPCG14-hybridoma 8 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val GlnPro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe ThrPhe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys ArgLeu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr ThrGlu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Phe Val Ser Arg Asp ThrSer Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala GluAsp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Val Tyr Gly Tyr Asp TyrTrp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr Val Thr Val SerSer 115 120 9 123 PRT Mouse VARIANT (1)...(123) HPCG11-hybridoma 9 GluVal Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Ile Thr Phe Ser Asp Phe 20 25 30Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45Ala Ala Ser Arg Asn Lys Ser Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 7580 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 9095 Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val 100105 110 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 10 123 PRTMouse VARIANT (1)...(123) HPCG132-hybridoma 10 Glu Val Lys Leu Val GluSer Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu SerCys Ala Thr Ser Gly Ile Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp ValArg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg AsnLys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly ArgPhe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu GlnMet Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala ArgAsp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly AlaGly Thr Thr Val Thr Val Ser Ser 115 120 11 123 PRT Mouse VARIANT(1)...(123) T15-myloma protein 11 Glu Val Lys Leu Val Glu Ser Gly GlyGly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala ThrSer Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln ProPro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala AsnAsp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile ValSer Arg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn AlaLeu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr TyrGly Ser Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr ThrVal Thr Val Ser Ser 115 120 12 123 PRT Mouse VARIANT (1)...(123)S63-myeloma protein 12 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly PheThr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly LysArg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr ThrThr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg AspThr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg AlaGlu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser SerTyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr Val Thr ValSer Ser 115 120 13 123 PRT Mouse VARIANT (1)...(123) Y5236-myelomaprotein 13 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe SerAsp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu GluTrp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu TyrSer Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser GlnSer Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp ThrAla Ile Tyr 85 90 95 Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp TyrPhe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115120 14 123 PRT Mouse VARIANT (1)...(123) S107-myeloma protein 14 Glu ValLys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 TyrMet Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 AlaAla Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 SerVal Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95Tyr Cys Ala Arg Asp Tyr Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp Val 100 105110 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 15 123 PRT MouseVARIANT (1)...(123) H8-myeloma protein 15 Glu Val Lys Leu Val Glu SerGly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser CysAla Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val ArgGln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn LysAla Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg PheIle Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln MetAsn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg AspTyr Tyr Gly Asn Ser Tyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala GlyThr Thr Val Thr Val Ser Ser 115 120 16 123 PRT Mouse VARIANT (1)...(123)M603-myeloma protein 16 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly PheThr Phe Ser Asp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly LysArg Leu Glu Trp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Gly Asn Lys Tyr ThrThr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg AspThr Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg AlaGlu Asp Thr Ala Ile Tyr 85 90 95 Tyr Cys Ala Arg Asn Tyr Tyr Gly Ser ThrTyr Trp Tyr Phe Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr Val Thr ValSer Ser 115 120 17 123 PRT Mouse VARIANT (1)...(123) W3207-myelomaprotein 17 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe SerAsp Phe 20 25 30 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu GluTrp Ile 35 40 45 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu TyrSer Ala 50 55 60 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser GlnSer Ile 65 70 75 80 Leu Tyr Phe Gln Met Asn Ala Leu Arg Ala Glu Asp ThrAla Ile Tyr 85 90 95 Tyr Cys Ala Arg Asn Tyr Tyr Lys Tyr Asp Leu Trp TyrVal Asp Val 100 105 110 Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115120 18 124 PRT Mouse VARIANT (1)...(123) M511-myeloma protein 18 Glu ValLys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 TyrMet Glu Trp Val Arg Gln Pro Ser Gly Lys Arg Leu Glu Trp Ile 35 40 45 AlaAla Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 55 60 SerVal Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 70 75 80Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 85 90 95Tyr Cys Ala Arg Asp Gly Asp Tyr Gly Ser Ser Tyr Trp Tyr Phe Asp 100 105110 Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 19 125 PRTMouse VARIANT (1)...(123) M167-myeloma protein 19 Glu Val Val Leu ValGlu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg LeuSer Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 25 30 Tyr Met Glu TrpVal Arg Gln Thr Pro Gly Lys Arg Leu Glu Trp Ile 35 40 45 Ala Ala Ser ArgSer Lys Ala His Asp Tyr Thr Arg Glu Tyr Ser Ala 50 55 60 Ser Val Lys GlyArg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Val 65 70 75 80 Leu Tyr LeuGln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Thr Tyr 85 90 95 Tyr Cys ThrArg Asp Ala Asp Tyr Gly Asn Ser Tyr Phe Gly Tyr Phe 100 105 110 Asp ValTrp Gly Ala Gly Thr Thr Val Thr Val Ser Ser 115 120 125 20 110 DNA Mouse20 ctcgagtcag gacctggcct cgtgaaacct tctcagtctc tgtctctcac ctgctctgtc 60actggctact ccatcaccag tgcttattac tggaactgga tccggcagtt 110 21 110 DNAMouse misc_feature (1)...(110) n=A,T,C, or G 21 ctcgagtctg ggcctnaactggcaaaacct ggggcctcag tgaagatgtc ctgcaaggct 60 tctggccaca ccttgactagttactggata cactgggtaa aanagaggcc 110 22 109 DNA Mouse misc_feature(1)...(109) n=A,T,C or G 22 ctcgagttct ggacctnagc tggtaaagcc tggggttcagtgaagatgtc ctgcaaggct 60 tctggataca ttcacnagct atgttataca ctgggtgaagcagaagcct 109 23 110 DNA Mouse 23 ctcgagtctg gacctgaact ggtaaagcctgggacttcag tgaagatgtc ctgcaaggct 60 tctggataca cattcaccag ctatgttatgcgctgggtga agcacaagcc 110 24 110 DNA Mouse misc_feature (1)...(110)n=A,T,C or G 24 ctcgagtcag gggctgaact ggtgaagcct ggggtttcag tgaagttgtcctgcaaggct 60 tctggctaca ccttcacnag ctactatatg tactgggtga agcagaggcc 11025 110 DNA Mouse misc_feature (1)...(110) n=A,T,C or G 25 ctcgagtctggggctaagct ggtaaggcct ggagcttnag tnaagctgtc ctgnagggct 60 tctggctactccttcacnag ctactggatg aactgggtga agcagaggcc 110 26 110 DNA Mousemisc_feature (1)...(110) n=A,T,C or G 26 ctcgagtctg gggctgagctggtgaggcct ggagcttcag tnaagctgtc ctgcaaggcc 60 tctcgtactc cttcaccagctcctgataac tgggtgaagc agaggcctgg 110 27 110 DNA Mouse misc_feature(1)...(110) n=A,T,C or G 27 ctcgagtcag gaggtggcct ggtgcagcct ggaggatccctgaaactctc ctgtgcagcc 60 tcaggattcg atttnagnag atactggatg aattgggtccggcagctcca 110 28 110 DNA Mouse misc_feature (1)...(110) n=A,T,C or G 28ctcgagtctg gaggtggcct ggtgcagcct ggaggatccc tgaatctccc ctgtgcagcc 60tcaggattcg atttnagnag ataatggatg agttgggttc ggcaggctcc 110 29 110 DNAMouse misc_feature (1)...(110) n= A,T,C or G 29 ctcgagtctg gaggtggcctggtgcagcct ggaggatccc tgaaagtctc ctgtgcagcc 60 tcaggattcg atttnagnagatactggatg agttgggtcc ggcagctcca 110 30 110 DNA Mouse misc_feature(1)...(110) n=A,T,C or G 30 ctcgcgtctg gaggtggcct ggtgcagcct ggaggatccctcaaactctc ctgtgcagcc 60 tcaggattcg atttnagnag atactggatg agttgggtccggcagctcca 110 31 110 DNA Mouse misc_feature (1)...(110) n=A,T,C or G 31ctcgagtcag gaggtggcct ggtgcagcct ggaggagccc tgaaactctc ctgtgcagcc 60tcaggattcg atttnagnag atactggatg agttgggtcc gcagctccag 110 32 110 DNAMouse misc_feature (1)...(110) n=A,T,C or G 32 ctcgagtctg ggggaggcttagtncagcct ggagggtccc ggaaactctc ctgtgcagcc 60 tctggattca ctttnagnagttttggaatg cactggattc gtcaggctcc 110 33 110 DNA Mouse misc_feature(1)...(110) n=A,T,C or G 33 ctcgagtctg ggggaggctt agtnnagcct ggagggtcccggaaactctc ctgtgcagcc 60 tctggattca ctttnagnag ctttggaatg cactgggttacgtcaggctc 110 34 110 DNA Mouse misc_feature (1)...(110) n=A,T,C or G 34ctcgagtcag gggctgaact ggtgaggcct gggcgttcag tnaagatgtc ctgcaaggct 60tcaggctatt ccttcaccag ctactggatg cactgggtga aacagaggcc 110 35 110 DNAMouse 35 ctcgagtcag gggctgaact ggcaaaacct ggggcctcag taaagatgtcctgcaaggct 60 tctggctaca cctcttcttc cttctggctg cactggataa aagaaggcct 11036 110 DNA Mouse misc_feature (1)...(110) n=A,T,C or G 36 ctcgagtctggacctnagct ggtgaagcct ggggttcagt taaaatatcc tgcaaggctt 60 ctggttactcattttctntc tactttgtga actgggtgat gcagagccat 110 37 110 DNA Mouse 37ctcgagtcag gggctgaact ggtgaagcct ggggttcagt aagttgtcct gaaggcttct 60ggctacacct tcaccggcta ctatatgtac tgggtgaagc agaggcctgg 110 38 91 DNAUnknown Synthetic 38 ggccgcaaat tctatttcaa ggagacagtc ataatgaaatacctattgcc tacggcagcc 60 gctggattgt tattactcgc tgcccaacca g 91 39 87 DNAUnknown Synthetic 39 cgtttaagat aaagttcctc tgtcagtatt actttatggataacggatgc cgtcggcgac 60 ctaacaataa tgagcgacgg gttggtc 87 40 82 DNAUnknown Synthetic 40 ccatggccca ggtgaaactg ctcgagattt ctagactagttacccgtacg acgttccgga 60 ctacggttct taatagaatt cg 82 41 86 DNA UnknownSynthetic 41 ggtaccgggt ccactttgac gagctctaaa gatctgatca atgggcatgctgcaaggcct 60 gatgccaaga attatcttaa gcaggt 86 42 91 DNA UnknownSynthetic 42 ggccgcaaat tctatttcaa ggagacagtc ataatgaaat acctattgcctacggcagcc 60 gctggattgt tattactcgc tgcccaacca g 91 43 87 DNA UnknownSynthetic 43 cgtttaagat aaagttcctc tgtcagtatt actttatgga taacggatgccgtcggcgac 60 ctaacaataa tgagcgacgg gttggtc 87 44 46 DNA UnknownSynthetic 44 ccatggccca ggtgaaactg ctcgagaatt ctagactagt taatag 46 45 50DNA Unknown Synthetic 45 ggtaccgggt ccactttgac gagctcttaa gatctgatcaattatcagct 50 46 131 DNA Unknown Synthetic 46 tgaattctaa actagtcgccaaggagacag tcataatgaa atacctattg cctacggcag 60 ccgctggatt gttattactcgctgcccaac cagccatggc cgagctcgtc agttctagag 120 ttaagcggcc g 131 47 131DNA Unknown Synthetic 47 tgaattctaa actagtcgcc aaggagacag tcataatgaaatacctattg cctacggcag 60 ccgctggatt gttattactc gctgcccaac cagccatggccgagctcgtc agttctagag 120 ttaagcggcc g 131 48 140 DNA Unknown Synthetic48 tcgaacttaa gatttgatca gcggttcctc tgtcagtatt actttatgga taacggatgc 60cgtcggcgac ctaacaataa tgagcgacgg gttggtcggt taccggctcg agcagtcaag 120atctcaattc gccggcagct 140 49 22 DNA Unknown Synthetic 49 aggtccagctgctcgagtct gg 22 50 22 DNA Unknown Synthetic 50 aggtgaaact tctcgagtca gg22 51 22 DNA Unknown Synthetic 51 aggtccagct gctcgagtct gg 22 52 22 DNAUnknown Synthetic 52 aggtccagct gctcgagtca gg 22 53 22 DNA UnknownSynthetic 53 aggtccagct tctcgagtct gg 22 54 22 DNA Unknown Synthetic 54aggtccagct tctcgagtca gg 22 55 22 DNA Unknown Synthetic 55 aggtccaactgctcgagtct gg 22 56 22 DNA Unknown Synthetic 56 aggtccaact gctcgagtca gg22 57 22 DNA Unknown Synthetic 57 aggtccaact tctcgagtct gg 22 58 22 DNAUnknown Synthetic 58 aggtccaact tctcgagtca gg 22 59 20 DNA UnknownSynthetic 5′ degenerate primer containing inosine at 4 degeneratepositions 59 aggtnnanct nctcgagtct 20 60 20 DNA Unknown Synthetic 5′degenerate primer containing inosine at 4 degenerate positions 60aggtnnanct nctcgagtca 20 61 19 DNA Unknown Synthetic 61 gcccaaggatgtgctcacc 19 62 39 DNA Unknown Synthetic 62 ctattagaat tcaacggtaacagtggtgcc ttggcccca 39 63 39 DNA Unknown Synthetic 63 ctattaactagtaacggtaa cagtggtgcc ttggcccca 39 64 19 DNA Unknown Synthetic 64ctcagtatgg tggttgtgc 19 65 26 DNA Unknown Synthetic 65 gctactagttttgatttcca ccttgg 26 66 23 DNA Unknown Synthetic 66 cagccatggccgacatccag atg 23 67 30 DNA Unknown Synthetic 67 aattttacta gtcaccttggtgctgctggc 30 68 39 DNA Unknown Synthetic 68 tatgcaacta gtacaaccacaatccctggg cacaatttt 39 69 32 DNA Unknown Synthetic 69 ccagttccgagctcgttgtg actcaggaat ct 32 70 32 DNA Unknown Synthetic 70 ccagttccgagctcgtgttg acgcagccgc cc 32 71 32 DNA Unknown Synthetic 71 ccagttccgagctcgtgctc acccagtctc ca 32 72 32 DNA Unknown Synthetic 72 ccagttccgcgctccagatg acccagtctc ca 32 73 32 DNA Unknown Synthetic 73 ccagatgtgagctcgtgatg acccagactc ca 32 74 32 DNA Unknown Synthetic 74 ccagatgtgagctcgtcatg acccagtctc ca 32 75 32 DNA Unknown Synthetic 75 ccagatgtgagctcttgatg acccaaactc aa 32 76 32 DNA Unknown Synthetic 76 ccagatgtgagctcgtgata acccaggatg aa 32 77 32 DNA Unknown Synthetic 77 gcagcattctagagtttcag ctccagcttg cc 32 78 33 DNA Unknown Synthetic 78 ccgccgtctagaacactcat tcctgttgaa gct 33 79 33 DNA Unknown Synthetic 79 ccgccgtctagaacattctg caggagacag act 33

What is claimed is:
 1. A composition comprising: a first genetic librarycontaining a plurality of diverse first polynucleotide sequences,wherein said sequences are carried by a first cloning vector, eachvector carrying a first polynucleotide sequence having a firstrestriction endonuclease site located proximal upstream to thetranslation initiation site of said first polynucleotide sequence, asecond genetic library containing a plurality of diverse secondpolynucleotide sequences, wherein said sequences are carried by a secondcloning vector, each vector carrying a second polynucleotide sequencehaving said restriction endonuclease site located proximal downstream tothe translation initiation site of said second polynucleotide sequence,wherein the first and second polynucleotide sequences encode a diversityof first and second proteins capable of forming a heterodimericreceptor.
 2. The composition of claim 1, wherein the first geneticlibrary comprises at least 10⁴ different sequences.
 3. The compositionof claim 1, wherein the second genetic library comprises at least 10⁴different sequences.
 4. The composition of claim 1, wherein the firstpolynucleotide sequences encode a functional portion of an antibodyheavy chain variable domains and the second polynucleotide sequencesencode a functional portion of antibody light chain variable domains. 5.The composition of claim 1, wherein said first and second types ofcloning vectors are lambda phage vectors.
 6. A composition comprising adiverse population of co-expression vectors obtained from recombinationof nucleic acid sequences comprising: a diversity of firstprotein-encoding sequences, which proteins are capable of forming aheterodimeric receptor with a diversity of second proteins, and adiversity of said second protein-encoding sequences, wherein said firstprotein-encoding sequences comprise a restriction endonucleaserecognition site proximal upstream of said sequences, and wherein saidsecond protein-encoding sequences comprise the same restrictionendonuclease recognition site proximal downstream of said sequences; andwherein said co-expression vectors each comprise a first- andsecond-protein encoding sequence joined by said restriction enzymerecognition site.
 7. The composition of claim 6, wherein the diversityof first protein-encoding sequences is at least 10⁴ different sequences.8. The composition of claim 6, wherein the diversity of secondprotein-encoding sequences is at least 10⁴ different sequences.
 9. Thecomposition of claim 6, wherein the first protein-encoding sequencesencode a functional portion of an antibody heavy chain variable domainand the second protein-encoding sequences encode a functional portion ofantibody light chain variable domain.
 10. The composition of claim 6,wherein said co-expression vectors are lambda phage vectors.